Biophotonics. Basic Microscopy. NPTEL Biophotonics 1

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1 Biophotonics Basic Microscopy NPTEL Biophotonics 1

2 Overview In this lecture you will learn Elements of a basic microscope Some imaging techniques Keywords: optical microscopy, microscope construction, microscopy techniques NPTEL Biophotonics 2

3 Overview of Optical Bio-imaging Optical imaging refers to the use of any of the optical contrast modes such as reflected or transmitted light, absorption, photoluminescence and so on. These modes are in contrast to other modes of imaging such as using x-rays or Magnetic Resonance Imaging (MRI) Unlike these other modes, optical radiation is not ionizing and therefore relatively less damaging to tissue. Furthermore they have better spatial and temporal resolution compared to, for e.g. MRI. The ability of optical imaging to probe vibrational spectrum of molecules makes it possible to do molecular specific imaging for different molecular species. The availability of compact light sources and detectors make it possible to have cost effective solutions for imaging and diagnostic applications. NPTEL Biophotonics 3

4 Basic Microscope Train The simplest image magnifier is a single lens. However to get uniform images free of distortions, one needs a more sophisticated design of the image train. 1) Ocular lens (eyepiece) (1) 2) Objective turret or Revolver or Revolving nose piece (to hold multiple objective lenses) 3) Objective 4) Focus wheel to move the stage (4 coarse adjustment, 5 fine adjustment) 5) Frame (6) 6) Light source, a light or a mirror (7) 7) Diaphragm and condenser lens (8) 8) Stage (9) NPTEL Biophotonics Image courtesy: Wikipedia Commons 4

5 Kohler Illumination Kohler illumination ensures uniform illumination across the sample plane. The illumination often comes from a lamp which contains a filament. Direct projection of the illumination onto the sample plane using a collector lens produces an image of the filament on the sample plane which is imaged by the objective lens along with the sample. Image courtesy: Wikipedia Commons NPTEL Biophotonics 5

6 Kohler Illumination In Kohler illumination, one puts a condenser in between the collector lens and the sample plane as shown in the figure below. As we see from the first image, the condenser does not focus the filament from the lamp onto the sample plane. One can defocus the filament image by using a diffuser which would also cut down the intensity of illumination. Kohler illumination avoids this problem. Image courtesy: Wikipedia Commons NPTEL Biophotonics 6

7 Numerical Aperture and Resolution Numerical aperture refers to the collection efficiency of the lens system. It is also related to the resolution of the image. Numerical aperture is defined as, NA = nsinθ, where n is the refractive index of the medium between the lens and focal plane The best resolution for a given wavelength, as we saw related to diffraction is 1.22λ/NA Increasing the numerical aperture enables the imaging of smaller features. NA can be increased by immersing the lens in high index fluids such as water or oil. This is the reason high magnification objectives are sometimes referred to as water immersion or oil immersion objective. θ NPTEL Biophotonics 7

8 Aberrations and Objectives We saw that refractive index is dependent on wavelength. This means that focusing, which depends on refraction, will also depend on wavelength. When one is imaging with white light, composed of multiple wavelengths, dispersion causes different wavelengths to have different focal planes. This aberration is called chromatic aberration as it results in colored streaks and fringes appearing in the microscopic image. Another aberration that occurs in microscopic systems is called spherical aberration. This happens because at large distances from the optic axis, the focal point of rays is shifted from those of paraxial rays. This results in distortion of features in the microscopic image. Modern microscopic objectives correct for these aberration by employing dispersion correcting lens pairs or triplets known as achromatic doublets/triplets which are compound lenses glued together. Similarly spherical aberration is corrected using aspherical lenses which have same focal plane independent of distance from axis. NPTEL Biophotonics 8

9 Upright and Inverted There are two major types of microscope frames which are used. They are called upright and inverted frames. In the upright microscope illumination is from the bottom (in the transmission mode) and the objective is placed at the top of the sample holder while in the inverted the objective is places below the sample holder and illumination is from the top. An inverted microscope provides more room for placing additional components near or in between the illumination and sample and generally the inverted frames have more flexibility in coupling additional light sources such as lasers into the microscope optical path. Imaging modes, such as phase contrast, DIC etc are possible with either frames. NPTEL Biophotonics 9

10 Dark-field Mode The common transmission or reflection imaging mode in a microscope is called bright-field because the background around the object of interest is brightly illuminated. There are several other imaging modes that increase the imaging contrast of several specimens. These are discussed in subsequent slides. The diagram below shows a dark-field imaging mode which is described in the next slide. Image courtesy: Wikipedia Commons NPTEL Biophotonics 10

11 Dark-field Imaging As the name suggests in the dark-field (DF) imaging mode, the background around the object is dark. This is done by using a field stop as shown in the diagram which blocks the central portion of the illumination while retaining the one at the edges. Image courtesy: Wikipedia Commons NPTEL Biophotonics 11

12 Dark-field Imaging Due to this special illumination, the background of the image is dark while the output essentially consists of the high frequency components of the image, i.e. edges of objects and so on which scatter light. Incidentally dark field illumination can be used to image metallic nanoparticles which have large scattering crosssection. The figure below shows a comparison of bright and darkfield images. Bright field (left) and dark-field (right) images of cellulose fibers image courtesy Wikipedia Commons NPTEL Biophotonics 12

13 Phase Contrast Microscopy Very often biological samples do not sufficient amplitude contrast, i.e. the reflectance or transmittance of various regions in the sample may be very similar in spite of structural variations, i.e. thickness may be different. In such cases one employs a phase contrast imaging mode where interference is used to differentiate between regions with slightly different refractive index or thickness. Recall that phase difference is determined by the optical path length which is the product of refractive index and geometric path length. This phase contrast microscope converts phase differences into intensity differences. The phase contrast technique was invented by Frits Zernike who was awarded the Nobel Prize for this work in NPTEL Biophotonics 13

14 Phase Contrast Microscopy The working principle of the phase contrast microscope relies on the fact for thin phase object the transmission is modified by T(x,y) = e jf(x,y) ~ 1 + jf(x,y), small phase modulation limit. This means that with respect to an unmodified light, the phase modulated light is 90 degrees out of phase (j represents a 90 degree shift). Phase contrast microscope creates regions which undergo phase modulation and regions that do not undergo phase modulation using a annular arrangement similar to dark-field microscopy. By providing an additional shift of 90 degrees to the phase modulated light and combining the two waves at the detector interference contrast between the phase modulated structure and the background is obtained. NPTEL Biophotonics 14

15 Phase Contrast Microscopy Phase contrast imaging allows one to study in-vivo behavior of systems in detail because otherwise one needs to use staining of cells with pigments and rely on differential absorption of the pigments by the different cell organelles. Some of the pigments may be toxic to the cells or may modify their behavior. Phase contrast image of cheek epithelial cells obtained without staining Image courtesy Wikipedia commons NPTEL Biophotonics 15

16 Phase Contrast Microscope The adjoining figure explains the construction of a phase contrast microscope. An annular illumination arrangement similar to dark-field microscopy is input using the phase ring. At the sample plane the most of the light goes through the unperturbed light path shown by the orange-yellow line. Some of the light passes through the sample and gets phase modulated. This is shown in the figure by the green line. Eye Piece Phase Plate Objective Sample Plane Condenser Phase Ring Phase Contrast Microscope NPTEL Biophotonics 16

17 Phase Contrast Microscope The objective focuses this light and passes through a a phase plate which matches the profile of the phase ring in such a way that the unperturbed light (orange line) is shifted by an additional 90 degrees and the intensity is also reduced to match that of the weaker phase modulated light (green line). At the eye piece the intensities of the perturbed (green) and unperturbed light almost matches, but with a phase difference of = 180. This generates a high contrast image of the phase modulation in the sample. Eye Piece Phase Plate Objective Sample Plane Condenser Phase Ring Phase Contrast Microscope NPTEL Biophotonics 17

18 DIC Imaging Another contrast mechanism for unstained, transparent samples is called Differential Interference Contrast or DIC. DIC images look similar to phase contrast images but the main difference is the use of polarized light in DIC. A DIC microscope is also called a Nomarski microscope from Georges Nomarski who developed this technique. In this technique a special prism called the Wollaston prism is used to create two co-propagating beams with orthogonal polarizations. Image courtesy: Wikipedia Commons NPTEL Biophotonics 18

19 DIC Imaging As their polarizations are orthogonal (i.e. E fields are pointing in orthogonal directions at each instant of time, they don t interfere as they propagate. The two orthogonally polarized beams go through the sample with a slight offset and therefore have a small phase difference with respect to each other. A second Wollaston prism recombines these two beams. At the second prism the polarization of the beams becomes equal causing them to interfere and convert the phase difference between them into an image contrast. But as the phase difference is produced from slightly different locations on the sample, the image is related to the phase gradient and acquires a psuedo 3D appearance which aids in image contrast. NPTEL Biophotonics 19

20 DIC Optical System The figure below shows the optical train of the DIC microscope. The Wollaston prisms produce co-propagating beams with orthogonal polarization which won t interfere. The beams are focused by the objective lens and recombined at the second Wollaston prism which equalizes the polarization of both beams, which now interfere producing the desired phase contrast. However, the recombination process happens in such a way that beams from slightly different positions are recombined causing an psuedo 3D appearance of the images. Image courtesy: Wikipedia Commons NPTEL Biophotonics 20

21 Polarization Microscopy As a final example we mention the use of polarized light for imaging samples that exhibit optical anisotropy and consequent changes in the polarization state of incident light. A general schematic of such a system is shown. For instance, in the system shown below, one may use a polarizer in the illumination path to create linearly polarized light which may undergo a rotation of polarization by some regions in the sample which may exhibit optical anisotropy. By placing a crossed polarizer prior to the detector, one can isolate the regions exhibiting optical anisotropy. Analyzer or Crossed Polarizer Sample Polarizer NPTEL Biophotonics 21

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