The light used in various applications can be either incoherent, for example, taken from a lamp or light emitting diodes or it can be from a coherent source such as a laser

1 Course: PG Paths hala-Biophysics

Paper 1: Foundations of Bio-Physics

Module 20: Applications of optics in biophysical instrumentation

Optics is a branch of science that studies light, its interaction with matter and control of light for applications. Optical instruments or machines are designed where light is used, guided and detected in a fashion that it provides solutions to specific problems in various branches of science and technology. An optical microscope is the most basic tool that is used everywhere by everyone dealing with light or optics. This device is used to see microscopic things at a magnified scale. Particularly, in biology and medicine, optical microscopes without a substitute are thought at the foremost to have them. Tissues, cells and structures are studied with the help of these microscopes. The light used in various applications can be either incoherent, for example, taken from a lamp or light emitting diodes or it can be from a coherent source such as a laser. Laser is another optical tool that has become so important in any field that everyone uses it in one form or the other in different kinds of applications. They are used in biology and medicine for scanning microscopy and imaging, tr eatment, surgery and diagnosis, etc.

Introduction

At the most, mirrors, lenses, glass fibers, prims and gratings are the optical elements which you would find in any optical instrument. We have already discussed these optical elements in the previous module. Here, we will take up a few optical instruments consisting of these elements and describe the application for which they are used in biology. We will start with optical microscopes first, explain how they work and present couple of examples where they have been used. We will talk about different variations of the optical microscope that are used in biological applications. We will go on to describe some of other most popular optica l instruments. These include phase contrast microscopy, dark field microscopy, optical coherence tomography, and Raman microscopy for molecular sensitive imaging. We will discuss some of the nonlinear optical microscopy techniques also. These include two photon excitation microscopy, second harmonic and third harmonic generation microscopy, nonlinear Raman microscopy. Most of these microscopy techniques are of scanning type, be it confocal microscopy or optical coherent tomography or the nonlinear optical microscopy and essentially used a laser as the light source for illuminating the specimen.

Objectives

In this chapter, we will learn about the following topics:

 Light microscopes

 Components of light microscopes

 Image reconstruction

 Fluorescence microscopy and confocal laser scanning microscopy

 Phase contrast microscopy

 Dark field microscopy

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 Optical coherence tomography

 Raman microscopy

 Nonlinear optical microscopy techniques

 Applications of lasers in surgery, therapy and treatment

1. Light microscopes

Optical microscope is an instrument that uses lenses or combination of lenses to produce magnified images of objects that are too small to be seen by naked eyes. Cells and tissues inside living bodies are tiny structures which are much smaller than the size human eyes can see normally. The purpose of light microscopes is to magnify and thereby study such objects or organisms. Light microscope works like a telescope except that the object is very close to the objective lens and very small. Light is sent through a path that first focuses the light into a tight beam and then passes that light through the sample. Subsequently the light transmitted through or reflected from the sample at the focus is sent through another lens or set of lenses depending on the desired magnification which helps in imaging the sample on the eye or a camera, typically a CCD (charged coupled device) camera to record an image. Light microscopes come in several forms some of which are discussed below.

1.1 Simple optical microscopes

Simple microscopes use a single lens to magnify an object as shown in Fig. 1. These microscopes can provide only limited magnification and hence limited optical resolution, i.e., the ability to distinguish between two adjacent objects. To achieve high magnifications, one needs to use sets of lenses as done in a compound microscope.

Fig.1: Optical block diagram of a simple optical microscope consisting of a single lens and a compound microscope that used combination of two lenses to produce image of a tiny object. Combination of lenses is useful for achieving desired magnification easily.

3 1.2 Compound light microscopes

Compound light microscopes use two sets of lenses, an objective lens and an eyepiece to produce image. Objective lens close to the object being viewed collects light and focuses a real image of the object inside the microscope. That image is then magnified by a second lens called eyepiece giving the viewer an enlarged and inverted virtual image of the object. Magnified image construction of a tiny object by compo und microscope is described in F ig. 1. The use of compound objective/eyepiece combination allows for much higher magnification. Each of the objective lens and the eyepiece can consist of two or more lenses. Compound microscopes are constructed in two forms called monocular and binocular microscopes. Monocular microscopes use one eyepiece while binocular microscopes use two eyepieces and hence help in reducing the eye strain. These microscopes feature a magnification power typically ranging from 40 X to 1400X.

2. Design and working of light microscopes

There are several components used to construct a light microscope. As a consequence, there can be many variants of the optical layout used in the construction of a microscope. Typically, an optical microscope looks like as shown in Fig. 2 where various components can be identified by the numbers marked on them. These components are, eyepieces or ocular lens, objective turret, a revolver or revolving nose piece where maximum three objective lenses can be mounted, objective lenses, Focus knob for course and, fine focusing adjustments, stage for placing the sample, light source for illuminating the sample, diaphragm and condenser for collecting the light transmitted through the sample and magnifying the image of the object, a mechanical stage for translating the sample across the focal point.

2.1 Components of an optical microscope

Typical list of optical components used in light microscopes has been summarized already in the above. A brief description is given here. Eyepiece or ocular lens: Eyepiece is a cylinder containing two or more lenses. Its function is to bring the image into focus for the eye. Typical magnification values for eyepiece are 2X, 10X and 50X. Objective turret: Objective turret is that part of the microscope which can hold a set of different magnification objective lenses. It allows the user to switch between those objective lenses. Objective lenses: In compound optical microscopes, there are one or more objective lenses that collect light from the sample placed at the focal plane of the objective lens. The objective lens assembly is usually in a cylinder form containing a glass singe or multi-element compound lens. Microscope objectives are characterized by two parameters namely, magnification and numerical aperture. The former typically ranges of magnification from 5X to 140X while numerical aperture ranges from 0.1 to 0.7, corresponding to focal length of about 40 to 2 mm, respectively. Objective lenses with higher magnification normally have a higher numerical aperture and shorter focal length. Focus knobs: Adjustment knobs move the stage up and down with separate adjustment for coarse and fine focusing. Stage: The stage is a platform below the objective which supports the specimen being viewed. In the center of stage is a hole through which light from below passes through to illuminate the specimen. The stage usually has arms to hold specimen. Light source: Most microscopes have their own adjustable and controllable light source. Condenser: The condenser is a lens designed to focus light from the illuminatio n source onto the sample. Mechanical stage:

A mechanical stage allows tiny movements of the sample holder via control knobs that reposition the sample as desired.

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Objective lenses are generally a combination of a few lenses and infinity corrected, i.e., parallel rays entering into the objective get focused in the focal plane. They are identified by two numbers printed on a commercially available objective lens, e.g., 0.2/40. The order of these two numbers can be company dependent. For the example here, the first number represents the numerical aperture which specifies the largest angle for light acceptance and the focal length.

The second number signifies the optical zoom that can be achieved with the objective lens.

Fig. 2: Typical look of an optical microscope. Shown here are various components of the microscope identified as numbers marked on them. See the text for details.

2.2 Image reconstruction

An object to be studied, for example a tiny biological organism so small that it looks like a dot, is put on a glass slide. The user looks through the microscope eyepiece. The amount of light illuminating the sample can be adjusted so that the image is seen clearly in the eyepiece. The sample stage is moved to a location where the object to be images is nearly in the middle of the field of view. Also, the stage can be moved up and down across the focal plane. This allows different layers of the object to be in focus of the objective lens and hence imaged on the eyepiece. A mirror at the bottom of the microscope reflects light rays up to the object through a hole in the stage. Objective lens magnifies the image which is made even larger when it is seen through eyepiece lenses. When we look at a typical animal cell with a light microscope it looks like as shown in Fig. 3. The cells are the basic building blocks of living organisms. They have got a very tiny nucleus surrounded by cytoplasm and plasma membrane at the periphery.

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Fig. 3: Composition and structure of a typical biological cell as seen from a high resolution optical microscope.

3. Fluorescence microscopy

A fluorescence microscope is much the same as a light microscope with added features to enhance its capabilities. In simple light microscopes one uses visible incoherent light from 400 to 700 nm for illuminating the sample. On the other hand, a fluorescence microscope, uses much higher intensity light, usually from a coherent source such as a laser producing light only in a small range of wavelengths. The light of specific wavelength excites one type of fluorescent specie in the sample of interest. This fluorescent specie after excitation emits light of a lower energy or a longer wavelength, usually in the visible range. Use of mercury and xenon arc lamps as high intensity illumination sources causes a large amount of excitation energy deposited in the specimen and a large number of fluorescent species possibly getting excited thereby providing those details in the image which can be accessed by using tradition light. The fluorescence light produces the magnified image of the object instead of original light source. Clearly, in fluorescence microscope, the sample we want to study is itself the light source.

Fluorescence microscope [6] is the most used microscope in the medical and biological field.

It is often used to image specific features of small specimens such as microbes. The typical structure of cells in animal bodies was shown in Fig. 3. There are different kinds of cells having different shape or size for they are specialized to perform a specific function inside body. An example is shown in Fig. 4 where nerve cells are shown in green (not true color, used for presentation only) as they appear in a fluorescence microscopy image of embryonic rat hippocampal neuron cells, cultured for 5 days and prepared for fluorescent staining [7].The image shows the spatial relationship between filamentous actin (red) and microtubule array (green) in the neurons. Actin staining (with a dye called rhodamine phalloidin) highlights the growing tips and filopodial extensions along axons and dendrites, while microtubule staining reveals the stable shafts of these processes.

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Fig.4: Fluorescence microscopy image of nerve cells. Image taken from Ref. 7.

In most cases, the sample to be imaged is labeled with a fluorescent substance known as a fluorophore. Typical optical layout of a fluorescence microscope is shown in Fig. 4. The specimen is illuminated through the objective lens with light taken from a broadband source. The excitation light is spectrally filtered using a wavelength filter for specific fluorophores. The illumination light is absorbed by the fluorophores (now attached with the sample) and causes them to emit a longer wavelength light. The same objective lens collects some of the fluorescent light from the sample which can be separated from the surrounding radiation and the excitation light by choosing a right dichroic mirror. As shown in Fig. 4, the dichroic mirror is such that it reflects the excitation light but allows transmission of the fluorescent light only. The filters are selected in such a manner that the microscope works only for specific excitation and fluorescent wavelengths thereby allowing the viewer to see only those regions in the specimen where the fluorophores are attached.

Fig. 5: Simple optical layout to explain the principal behind the fluorescent microscopy.

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In the excitation and light emission processes undergoing in the fluorophores, electronic transitions between various energy levels take place. While returning from the excited energy states, the electrons jump to those states with certain probability whic h are radiative, i.e., the energy relaxation process produces light. The objective lens collects the fluorescent light thus produced. This fluorescent light passes through the dichroic mirror and is sent to the eyepiece or a multi-pixel photodetector to produce an image of the sample.

4. Confocal laser scanning microscopy

Laser scanning confocal microscopy [8] or simply confocal microscopy uses the principle of fluorescence excitation to produce high resolution blur- free images of thick specimens with depth selectivity three-dimensional (3-D) imaging. This is achieved with the help of a pin hole placed at a given point in the excitation beam path that helps select light coming from only the focused region in the sample. A conventional microscope sees as far into the specimen as the light can penetrate, while a confocal microscope only sees images one depth level at a time. This is useful for surface profiling for opaque specimen, while for non-opaque specimen, interior structures can be imaged. For interior imaging, the quality of images is greatly enhanced over simple microscopy because image information from multiple depths in the specimen is not mixed or superimposed and the resolution along both depth and cross-section is better. Another important point to note here is that in confocal microscopy images are taken point-by-point and reconstructed with a computer, rather than projected through an eyepiece. In biology, confocal microscopes are used to investigate the structural properties of cells and the location of particular structures or protein populations within those cells in fixed tissues. The movement of biological entities in live cells such as vesicles or even individual proteins, can also be studied.

Typical optical set up for confocal microscopy is shown in Fig. 5 where a laser (blue, for example) is used to provide the excitation light. The laser beam reflects off a dichroic mirror and hits two scanning mirrors mounted on motorized mirror mounts; these mirrors scan the laser beam across the sample. In the example shown here, red light is emitted from the fluorescing sample from each point of excitation. This emitted light is guided back through the dichroic mirror (allows it completely) and to a photodetector, usually a photomultiplier tube.

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Fig. 6: Typical optical layout of a confocal microscope.

Point to be noted, in confocal microscopy, only one point of the sample is observed at a time but never a complete image of the sample. The detector is attached to a computer which builds up the image, one pixel at a time for every point on the sample during scanning of the laser beam.

Pinhole in the beam path removes unwanted, out of focus fluorescence, giving an optical slice of a 3-D image constructued on the computer. The pinhole blocks the passage of any out-of- focus light or stray light into the detector. This means that the only light entering the detector comes from near the focal plane of the objective lens of the microscope. With the help of the scanning mirrors the focused beam on the sample is taken across an area of the sample thus producing an image that is a slice through the object and surrounding material. This is known as optical slicing by which one is able to see inside the object of interest.

5. Phase contrast microscopy

When light waves travel through a medium other than vacuum, interaction with the medium causes the wave amplitude and phase to change in a manner dependent on properties of the medium. Changes in amplitude (brightness) arise from the scattering and absorption of light, which is often wavelength dependent and may give rise to change in color of the light that was sent in. Photographic equipments and the human eyes are only sensitive to the amplitude variations. Without special arrangements phase changes are invisible. For transparent specimens, phase contrast microscopy is used. It is widely used for examining such specimens in biological systems. The phase contrast microscope is able to show components in cells or bacteria, which would be very difficult to see them in an ordinary light microscope or bright field microscope.

Essentially, in phase contrast microscopy [9], phase shifts introduced in the light passing through transparent specimens is converted into brightness changes in the image. The light passing through a transparent part of the specimen travels slower due to which it is phase shifted as compared to the incident light. This difference in phase is not visible to human eye. However,

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the change in phase can be increased to half a wavelength by a using a phase-plate in the phase contrast microscope and thereby causing a difference in brightness. This makes the transparent regions in the sample shine out in contrast to its surrounding.

An example of phase contrast microscopy is presented in Fig. 9 where results from both bright field normal microscope and a phase contrast microscope are shown for cells from human glial brain tissues grown in monolayer culture bathed with a nutrient medium containing amino acids, vitamins, mineral salts and fetal calf serum [9]. In bright field microscopy images (left images in Fig. 9), the cells appear semi-transparent and only some regions a partially visible.

However, in the phase contrast microscopy image (right side image in Fig. 9), more structural details are observed.

Fig. 7: Living cell image in bright field and phase contrast microscopy. Images taken from Ref. 9.

The basic pr inc iple behind the phase microscopy to work, make phase changes due to transparent objects vis ib le in ter ms o f giving r ise to co ntrast c hange is to separate the illuminat ing background light fro m the spec ime n scattered light whic h make up the foreground details, a nd to ma nip ulate t hese differe nt ly. The work ing pr inc ip le of a phase contrast mic roscope is exp la ined in Fig. 10 (taken fro m Re f. 10). In this, a r ing shaped illuminat ing light (shown b y green co lor in F ig. 10) that passes a condenser annulus is foc used on t he specime n by a conde nser lens. So me of the illumina t ing light is scattered by the specime n (shown in ye llow) and t he rema ining is una ffected by the specimen whic h for ms the background light s hown in red. Whe n observing unsta ined bio logica l spec ime ns, the scattered light is weak and typ ica lly phase shifted by - 90°

relat ive to the background light. This results into t he fore ground (blue vector) and the background (red vector) having near ly t he sa me intens ity a nd he nce a low image contrast (see t he descr ipt ion (a) provided in the le ft co lumn in F ig. 10). Now the contrast in t he image is improved in two steps. The background light is p hase shifted by - 90° by passing it t hrough a phase s hift r ing. This e limina tes t he phase d iffere nce between t he background and t he scattered light, lead ing to an increased intens ity

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differe nce between fore ground and background (descript io n (b) in F ig. 10). To fur ther increase the contrast, t he background light is d immed fur ther b y use o f a gra y filter ring (descript io n (c) in Fig. 10). By doing so some of the scattered light will be phase shifted and dimmed by the r ings. However, the background light is a ffected to a much greater exte nt thereby creat ing a nice phase contrast e ffect. The who le of the above discuss io n is for negat ive phase contrast. In a similar manner, one can make slight changes in t he opt ical arra nge me nts in t he setup shown in Fig. 10 for ge nerat ing posit ive phase contrast. In t he later, t he backgro und light is phase s hifted by +90°

whic h as a result will be 180° out o f p hase relat ive to t he scattered light. This means that t he scattered light will be subtracted now fro m t he background light in descr ipt ion (b) in Fig. 10 to form a n ima ge where the foregro und is darker than t he background.

Fig. 8: Working pr inc ip le of s p hase contrast microscope. The ima ge taken fro m Ref. [10].

6. Dark fie ld micros copy

Dark fie ld microscopy [11] is a specia l arra nge ment of opt ics where unsta ined sa mp les are illuminated in s uch a wa y that t he y appear bright aga inst a dark background. As shown in Fig. 11, a dark fie ld microscope contains a spec ia l condenser t hat is designed to for m a ho llow cone o f light fro m light inc ident o n it. This he lps in scattering of light caus ing it to re fract o ff fro m t he specime n under observat ion at a part ic ular angle. The light at t he apex o f t he light cone is foc used at t he p la ne o f t he specime n, as t his light mo ves past the specime n p lane it spread s again into a ho llow cone. The objective lens sits in the dark hollo w region o f this light cone. A lt hough the light trave ls around and past the objective le ns, no rays enter it. The ent ire fie ld appears dark when there is no

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specimen o n t he microscope samp le sta ge, a reason behind the na me dark fie ld microscopy. W hen there is a specime n o n t he microscope samp le s tage, t he light at the apex of the cone str ikes it. The light ra ys part ia lly get scattered by the sa mp le suc h that they are captured by t he objective le ns which create an ima ge at the eyep iece.

Fig. 9: Optical arrange me nt in a darkfie ld opt ica l microscope where the light for illuminat io n o f the specime n enters fro m botto m.

Specime ns to be observed under a dark fie ld microscope should be prepared carefully s ince dust and other part ic les also scatter the light a nd are easily detected or ima ged at the eyep iece. Dark fie ld microscopy has many applicat io ns in microbio lo gy.

It allows t he vis ua lizat ion o f live bacter ia , and he lps d ist inguis h so me struct ures s uch as rods, curved rods, spirals, etc. agains t t he move me nt of t he bacteria. I mages s hown in F ig. 12 are of a deer t ick taken us ing a br ight fie ld nor ma l microscope ( left ima ge in the figure) vs that taken us ing a dark fie ld microscope, taken from re f. [12].

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Fig. 10: Live bacter ia ima ge as it appears under a br ight fie ld a nd a dark fie ld microscope, respective ly. Ima ge taken fro m open source [12].

7. Optical cohe re nce tomography

Optical co herence to mograp hy (OCT) [13] is anot her t ype of opt ica l ima ging by light beam scanning technique t hat is used for noninvas ive ima ging app licat ions in b io lo gy and med ic ine. OCT delivers higher resolut io n t han ot her k ind sound or radio freque ncy based devices in med ical fie ld because the light used in OCT has muc h s ma ller wave lengt h. OCT can capt ure microscopic regio ns a nd create 3- D view fro m within bio logica l t iss ues. In opht ha lmo logy OCT has been ver y s uccessful provid ing high resolut ion vo lume images, cross- sectiona l ima ges of t he ret ina, retina l nerve fiber layer and the optic nerve head [14].

Only a s ma ll port io n of the light inc ident on t he b io logica l t iss ues gets re flected back while most o f t he light gets scattered off at large a nge ls. I n convent io na l ima ging, this d iffuse ly scattered light contr ibutes to t he unwanted background t hat b lurs the ima ge. However, OCT is based on low cohere nce inter ferometr y whic h means o nly that light is collected whic h has tra ve lled a certain pat h lengt h and a ll t he rest of t he light is rejected. Typica lly, infrared light is used whic h can penetrate into the scatter ing med ium much deeper tha n t he vis ib le light. Sche mat ic of a t yp ica l OCT setup is s hown in F ig. 13. Light fro m t he source is d ivided into two ar ms by us ing a beam- splitter. The arm conta ining t he samp le is ca lled the sa mp le ar m and t he other having s imp ly a mirror to reflect back the light is called re ference ar m. The comb inat io n of re flected lights fro m the t wo ar ms creates an inter fere nce pattern o n t he photodetector placed on the opposite s ide o f the re ference ar m in Fig. 13. The inter fere nce pattern deve lops only whe n the light fro m both t he ar ms has trave led the sa me path le ngt h and he nce acquire the sa me p hase. By scanning the mirror in t he re ference ar m, a depth pro file in the sa mp le is obta ined for t hat point on t he sa mp le whic h has been illuminated. Note that t he regions in the sa mp le whic h can re flect more light will create greater contrast in t he inter fere nce pattern t ha n rest o f t he re gions in t he sa mple. Also, any light t hat is outs ide t he short co herence will not inter fere. This k ind o f re flect ivit y pro file obta ined by scanning the light beam alo ng t he axia l direct ion is called an A- scan. Clearly, A- scan conta ins t he infor mat io n about t he spat ia l d ime ns ions a nd locatio ns o f str uctures within t he samp le. Now to achieve cross - sectiona l view or to mograph o f the sa mp le, the light beam is scanned on t he sa mp le latera lly wit h the he lp of ga lvano metr ic

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scanners creat ing B- scans. Together t he A- and B- scans provide the vo lume tr ic vie w of the three- dime ns io na l struct ura l infor mat ion about t he sa mp le.

Fig. 11: Optical la yout of an opt ica l coherenc e to mograp hy (OCT) setup.

There are ma ny var iants o f t he OCT setup t hese days wit h one or more cha nges made in t he opt ica l setups fro m t hat shown in F ig. 13 . Each one o f t hese is comparat ive ly better t ha n t he other for ac hie ving a spec ific req uire ment in t he ima ging of the sa mp le. Primar ily, these optica l arrange me nts are fro m two categor ies, i.e., eit her t ime- doma in or t he Four ier do ma in OCT. Time - doma in OCT (TDOCT) is more or less t he sa me as d isc ussed above and is the de vice used curre nt ly in opht ha lmo lo gy.

High qua lit y images req uire longer t ime to create t he m. Therefore t ime is the ma jor limitat io n of t his techniq ue. This is better suited for uses where deeper features are to be seen but wit h low s igna l to no ise rat io. In t he Four ier - doma in OCT (FDOCT), one does not require a scanning mirror in t he re ference ar m t hereby mak ing t he technique much faster, at least 10 times fas ter tha n t he t ime - doma in OCT. This technique re lies on the pr inc ip le o f spectral inter fero metr y a nd the Four ier trans for m t hat ca n be done online at ver y high speeds. Optica l s igna ls are collected from t he sa mp le as a funct ion of the co lor (wave le ngth) o f t he scattered light. Spectral doma in OCT (SDOCT) is a partic ular imp le me ntat io n o f FDOCT that collects a ll o f t he wa ve le ngths o f the

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scattered light at t he sa me t ime us ing a specia lly designed spectrometer. The inter fero metr ic s igna ls are detected as a funct io n o f the opt ica l freque nc ies allo wing ima ging speeds that are about 50 times fast t hat TDOCT and provide greater number of ima ges per unit area. For examp le, in ret ina l t iss ue ima ging, high reso lut ion ima ges can be acquired by high dens it y raster scanning wit h minima l art ifacts ar is ing fro m eye mo ve ments.

8. Raman s pe ctros copy and Raman imaging

Based on Ra man scatter ing [15], i.e., light scattered fro m a samp le is freq uenc y shifted by an amo unt that is character ist ic to the mo lecular rotat ion a nd vibrat iona l freque nc ies, Ra ma n spectroscopy provides fingerpr ints o f cells, t issues or b io fluids by whic h t he const it ue nt mo lecules ca n be ide nt ified. More details about t he techniq ue are beyond t he scope of t he curre nt module a nd hence, interested readers can go thro ugh the re ferences pro vided below. Essent ia lly, a laser source provid ing inte nse light at a given wave le ngt h is used to opt ica l exc ite t he sa mp le under invest igat ion. Scattered light is filtered us ing appropriate opt ica l filters and se nt to a spectrometer which disperses the light onto a CCD (charge coupled device) detector.

Rama n ima ging o f a sa mp le is not hing b ut tak ing laser scanned ima ge of t he sa mple for a partic ular optica l freque ncy t hat is character ist ic to t he samp le called t he Ra man freque nc y. The basic pr inc ip le behind Ra ma n scatter ing and spectroscopy is exp la ined brie fly in F ig. 16. Light scatter ing fro m a mo lecule res ults into Rayle igh scattered photons ( no cha nge in freque ncy) and Ra man scattered p hotons ( freq uenc y s hifted by the vibrat io na l freque nc ies of the mo lec ule). The ener gy leve l d iagra m a nd the optica l trans it io ns tak ing place in t he e xc itat io n and scatter ing processes are also s hown in the respective co lors. Light scatter ing fro m a mo lec ule res ult ing into Ra yle igh scattered photons ( no cha nge in freque ncy) and Ra man scattered p hotons ( freq uenc y s hifted by the vibrat io na l freque nc ies of the mo lec ule). The ener gy leve l d iagra m a nd the optica l trans it io ns tak ing place in t he e xc itat io n and scatter ing processes are also s hown in the respective co lors. The inc ident laser photo n mo mentar ily pro motes the s yste m into a virt ua l state fro m whic h t hey sponta neous ly la nd into o ne of t he vibrat iona l le ve ls of the s yste m or ret ur n back to the sa me gro und state as the init ia l o ne. The overa ll Rama n spectrum c haracteris t ic to the samp le looks like that is s hown here. It conta ins ma ny spectra l lines each of w hic h is a Ra ma n trans it ion a llowed in the sa mp le.

Once the Ra man freque ncy is opt ica lly se lected by us ing o ne or a set o f optica l filters, the opt ical setup for Ra ma n ima ging is sa me as t he o ne used for fluorescence microscopy disc ussed above. The collect ion o f the light t hat is scattered from the samp le is taken in a perpend ic ular d irect ion fro m t he d irect ion of exc itat io n a nd t his is roughly the only d ifference between t he fluorescence microscopy and the Ra man microscopy. F luorescence spectroscopy probes more o f t he e lectronic states whereas Rama n spectroscopy probes the vibrat iona l states o f t he syste m. Howe ver, Ra man scattering process is more t ha n s ix orders o f ma gnit ude weaker t han the fluorescence and consequent ly could not be observed microscopica lly in human ce lls unt il recent ly.

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Because of t his limitat io n, tho ugh mo lec ular state dependent a nd he nce use ful for ear ly disease detection a nima ls, Ra ma n spectroscopy was not successful late ly.

Fig. 12: Light scatter ing fro m a mo lec ule res ult ing into Ra yle igh scattered photons (no cha nge in frequenc y) and Ra ma n scattered photons (freque ncy shifted by t he vibrat io na l freq uenc ies o f the mo lecule ). The ener gy leve l diagra m and t he optica l tra ns it io ns tak ing p lace in the e xc itat io n and scatter ing processes are also shown in t he respective co lors. The o vera ll Ra ma n spectrum characteris t ic to the sa mple looks like t hat is s hown here. It conta ins ma ny spectral lines each o f which a Ra ma n trans it ion poss ible in t he samp le.

Recent deve lop ments in t he laser techno logy b y whic h high peak power laser pulses are availab le, other for ms o f Ra man scatter ing mec hanis ms suc h as coherent ant i- Stokes Raman scatter ing, st imula ted Ra man scatter ing, s ur face enha nced Ra man scattering ha ve got q uite a success because of ma ny order higher s igna l to no ise rat io as compared to spontaneous Ra man scatter ing a nd high speed of data collect io n. This has made it possib le to apply t he Ra ma n spectroscopy and microscopy for ima ging applicat ions in b io logy a nd med ic ine. To de monstrate t he power o f Ra ma n mic roscopy in b io logy, F ig. 17 compares a bright fie ld microscopy ima ge and Ra man mic roscopy ima ge o f t he brain t iss ue o f a mouse 24 days a fter imp la ntat io n wit h a human gliob lasto ma mult ifor me xe nogra ft. Clear ly, over who le o f t he re gion, the t iss ues appear to be norma l in t he br ight fie ld ima ge. On t he ot her ha nd, wit h st imulated Rama n scatter ing (SRS) microscopy, t umor t issue is de lineated eas ily by t he higher nuc lear- to- cytoplas mic rat io. This has been taken fro m re fere nce [16].

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Fig. 13: Comparis io n o f a br ight fie ld microscopy ima ge o f a mouse bra in with stimulated Ra ma n scatter ing (SRS) mic roscopy. Das hed line in t he SRS images ind icates t he t umor mar gin. SRS ima ges represent a linear co mb inat io n o f symetr ic streaching of C - H Raman s igna ls at 2845 and 2930 wave numbers.

Taken from Re f. 16.

9. Nonline ar optical mic ros copy and imagi ng te chniques

Nonlinear opt ical ima ging techniques [17] re ly on the s trengt h o f t he s igna ls which are generated by e lectro nic/ato mic or vibrat io na l tra ns it io ns between correspond ing e ner gy le ve ls that are ind uced by simulta neous absorptio n of more tha n one inc ident photo ns.

There ca n be many poss ib ilit ies o f t he nonlinear interact io ns tak ing p lace in a ny specimen e xc ited by light fro m a pulsed laser . A few o f these are presented in F ig. 19 where t he sa mple is e xc ited by red laser light represented by red arrows a nd waves a nd fie ld Ein(). The light produced in t he second har monic ge nerat ion (SHG), i.e., freque nc y doub le o f t he inc ident, third har mo nic generat io n ( THG), i.e., freque ncy trip le o f t he inc ident and two - photon exc ited fluorescence (TPEF), i.e., freq uenc y of the fluorescence being s ma ller t ha n doub le o f t he indent o ne, are also s hown in F ig. 19 us ing d ifferent co lors, arrows a nd wa ves. In t he figure, hor izo nta l lines represent the specific ener gy le ve ls of t he syste m under study.

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Fig. 14: Schemat ic ener gy le ve l dia gra m o f t he no nlinear opt ical processes under go ing in t he second har mo nic ge nerat ion (SHG), third har monic ge nerat ion (THG) and two- photon exc ited fluorescence (TPEF). The inc ident light is represented as Ein() having freque ncy  and the light ge nerated in the var ious nonlinear processes is a lso s hown us ing t he respect ive fie lds a nd freque nc ies.

The d iffere nce in t he absorptio n a nd emiss io n processes has been highlighted by us ing d iffere nt colors for each of t he m.

Since, the p hotons ge nerated in t he no nlinear processes have freque ncy d ifferent than t he inc ide nt ones it’s easy to spectrally separate the m by us ing optica l filters and dichroic mirrors. Genera lly, the nonlinear opt ica l s igna l st rengt h is quite weak and hence high ga in p hotodetectors such as p hotomult ip lier t ubes (PMTs) are used. The design o f the no nlinear optica l microscope is not diffe rent fro m t he other microscopes discussed above suc h as t he fluorescence or the Ra ma n microscope. The only differe nce is in t he laser so urce t hat is used for e xc itat io n o f t he sa mp le. The laser source sho uld be ab le to provide high peak power laser pulses so that t he e ffic ie ncy of new freq uenc y ge nerat ion in t he nonlinear processes is s ignifica nt for detectio n. As shown in F ig. 20, laser pulses fro m t he laser source are focused onto t he sa mple b y use of a n object ive le ns a nd t he back scattered light t hat was generated t hro ugh the nonlinear processes is spectrally separated by us ing d ichro ic mirrors and filters fro m the inc ide nt light and is d irected to a PMT for detection. The sa mp le is p laced on a scanning sta ge and t he objected is mo unted on a z- scanner so t hat full 3- D ima ges of the sa mp le ca n be recorded. For each point o f e xc itat ion on t he sa mp le, t he s igna l is saved and p lotted us ing a co mp uter. The who le s yste m is co mp uter auto mated, i.e., the samp le sca nning a nd ima ge creat io n processes are so fast t hat rea l t ime imaging o f the samp le can be achieved easily.

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Fig. 15: Schemat ic o f a no nlinear optica l microscopy a nd imaging set up. Laser beam in the for m o f e nergy pulses are taken from a p ulsed laser and scanned point- by- point on a sa mp le. The scattered light is co llected in t he re flect ion geome try ( it can be done in t he tra ns miss io n mode also) a nd detected us ing eit her a PMT or a spectro meter. W ide - fie ld ima ging o f the sa mple can a lso be done in the sa me setup by illuminat ing t he samp le wit h white light. Var ious filter and dic hro ic mirrors are used to spectrally se lect li ght in t he interested wave lengt h ra nge.

Note that higher t he input intens it y, higher will be t he stre ngth o f t he nonlinear optica l signa l. There fore, the laser beams are focused onto the samp le and the nonlinear s igna ls are ge nerated and co llected only fro m t he re gions wit hin t he focal vo lume o f the laser in t he samp le. This partic ular fact allows sect ioning in ima ging of a bulk sa mp le a nd to acquire high reso lut io n 3 - D view o f it. As an exa mp le of nonlinear opt ical mic roscopy and ima ging of b io logica l s yste ms, Fig. 21 shows representative TP EF/SHG ima ges of co llage n bund les and e last ic fibers in nor ma l huma n skin dermis [18]. The SHG image br ings o ut t he s truct ura l morp ho logy o f the highly ordered crysta lline co lla gen fibr ils while TPEF is recorded fro m t he e last ic fibers which are much less respons ive in t he SHG ima ging.

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Fig. 16: Nonlinear optica l microscopy of huma n skin. Representat ive SHG (a) and TPEF (b) ima ges of colla ge n bund les and elast ic fibers in nor ma l sk in dermis. (c) Over la y o f t he SHG/ TPEF images. Sca le bar is 20 m. Taken fro m Ref. 18.

10. Lase r s urge ry

Fina lly, we would like po int o ut here that lasers ha ve found huge number of applicat ions in med ica l scie nce. Not only for ima ging applicat ions, lasers are being used for s ur ger ies and t herap ies as we ll. For exa mp le, LASIC and LASEK are the sur gica l met hods used for laser eye sur gery for correcting the re fract ive errors in e yes [19]. Laser eye surgery was made possib le in t he 1980s after researchers at the IMB could inc ise a nima l t issue prec ise ly wit ho ut leaving scar t iss ue by us ing a n exc imer laser. In ge nera l, laser therap ies are med ica l treat ments t hat use foc used laser light at specific wave le ngt h for cutt ing and ab lat ion o f t iss ues very prec ise ly in t he se lected region only wit hout da ma ging surro und ing t issues. Curre nt ly, laser t herapy is used in ma ny procedures suc h as for shr ink ing or destruct io n o f t umors, polyps, or precancerous growt hs, removing k idne y stones, treatme nt of cancero us cells and so on [20].

Summary

1. Optical microscope is an instrument that is used to see objects difficult to observe with naked eyes. In this module, we have discussed about the components and working of optical microscopes.

2. Optical microscopes can be found in many variants. It all depends on the components and optical layout that is used to construct the microscope.

3. Depending on the requirements, one can modify the existing optical microscope to perform either bright field microscopy, dark field microscopy, fluorescence microscopy or phase contrast microscopy.

4. Laser scanning confocal microscope is a special form of fluorescence microscope where a laser is used to optical excite the sample under observation and a pin- hole placed in the

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excitation path ensures that light coming from only the focal point reaches the camera. This configuration is highly useful for obtaining high resolution 3-D volume images of the biological samples.

5. Raman microscope is similar to fluorescence microscope in construction but it has the advantage that one can obtain molecular vibration specific images. Therefore, in detections of cancer or other disease, Raman microscopy is highly useful.

6. Optical coherence tomography is one of the most popular technique in ophthalmology where one can create 3-D scanning images at high speeds.

7. Using high power lasers, there are nonlinear optical microscope which are designed to bring different types of contrast mechanism in the sample imaging.

8. Pulse lasers are the light sources that are in demand these days because they are highly useful in surgery, therapy and treatment.