for courses SK2500 & SK2501, Physics of Biomedical Microscopy, Physics of Biomedical Microscopy, Extended Course Kjell Carlsson

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1 ? Biomedical & X-ray Physics Kjell Carlsson Problems (and solutions) for courses SK500 & SK501, Physics of Biomedical Microscopy, Physics of Biomedical Microscopy, Extended Course by Kjell Carlsson Physics Dept., KTH, Stockholm, 008

2 Note: This little collection of problems was originally compiled in the academic year 003/004 when the course Physics of Biomedical Microscopy was given for the first time, and therefore no previous written examinations existed. Such previous examinations are usually of considerable interest to students, and therefore the following problems were extracted from examinations given in a previous course (that was similar, but not identical, to the current course). The problems are mixed, meaning that they are not arranged in the chronological order of the course content. There is also a strong bias toward the parts covered in the compendium Imaging Physics and the confocal part of Light Microscopy. In order to cover some of the new stuff in the present course, such as Köhler illumination and DIC, problems 1 and were added (these are not old examination problems).

3 3 Problem 1 Köhler illumination is much used in microscopy. Look at Fig. 4 in the compendium Light Microscopy and answer the following questions. a) When the luminous field diaphragm is closed down, less light from the lamp will reach the specimen. Does this mean that the illuminance level in the specimen is reduced? Explain why, or why not, this is the case. b) In photography, closing down the diaphragm opening of the camera lens will increase the depth of field. Which of the two diaphragms in a microscope will affect the depth of field, and why doesn t the other one have the same effect? c) Which of the two microscope diaphragms affects the resolution, and how should it be adjusted for best results? Why doesn t the other diaphragm affect the resolution? d) Sometimes people with eyeglasses cannot get close enough to the eyepiece of the microscope. What is the effect if the pupil of the eye is placed too far away from the eyepiece, and why? Problem A DIC microscope is set up in the usual way, i.e. with a phase shift ϕ = π between the sheared beams for equal specimen pathlengths, see Light Microscopy page 8. The light intensity obtained when the ϕ two sheared beams are recombined is given by the function I = I cos 0. Thus, for the case of I equal pathlengths, we get I = 0, or 50% of maximum intensity. To clearly distinguish a change in intensity level, we require a change of, say, 0% (i.e., I becomes 40% or 60% of maximum level). Under these circumstances, what is thickness, d, of the thinnest plane-parallel piece of glass that can be detected when inserted into a DIC microscope according to the figure? We assume that monochromatic light with λ = 550 nm is used. Air, n = 1 d Glass, n = 1.5 Two beams in the DIC microscope sheared by the distance δ. δ Problem 3 True or false, and why? (Consider incoherent imaging) If we know what the point spread function of an optical system looks like, we know everything there is to know about the imaging properties of the system. If we know what the point spread function of a diffraction-limited optical system looks like, we know everything about the imaging properties of the system. If we know the Rayleigh resolution limit of an imaging system, we know everything about the imaging properties. If we know the Rayleigh resolution limit of a diffraction-limited imaging system, we know everything about the imaging properties.

4 4 Problem 4 Let us define volume resolution in confocal microscopy as the product of the resolution figures in the x, y and z directions. (If, for example, the resolution figures are 1.0 μm, 0.5 μm and 1.5 μm, the volume resolution would be = μm 3.) a) What is the mathematical relationship between the wavelength of the light and the volume resolution in confocal fluorescence microscopy? b) What is the mathematical relationship between the numerical aperture of the objective and the volume resolution in confocal fluorescence microscopy? (It s okay to assume that the numerical aperture is small in order to simplify the calculations) For simplicity you can assume that the excitation and fluorescence wavelengths are equal. You are free to select which measure of resolution you want from the ones presented in the course. Problem 5 In this problem you are asked to select a suitable dichroic beam-splitter and barrier filter for confocal fluorescence microscopy. A simplified schematic drawing of the set-up is shown in the figure below. Objective Dichroic beam-splitter Aperture Barrier filter Detector Fluorescent specimen λ exc = 488 nm The emission spectrum of the fluorophore used is given in the figure below. Intensity λ (nm)

5 5 You have a choice of 4 different dichroic beam-splitters, whose transmission curves are shown below. Note that in all cases light that is not transmitted is reflected. 90% Transmission a b c d 5% λ (nm) You also have a selection of different barrier filters, whose transmission curves are given in the figure below. 95% Transmission % λ (nm) Since the light intensity is very low, it is important that as many fluorescence photons as possible can reach the detector. However, it is also important that absolutely no laser light (which is many orders of magnitude stronger than the fluorescent light!) can reach the detector. Select a suitable dichroic beamsplitter (a-d) and barrier filter (1-6) to give the best results under the circumstances. Explain carefully how you make your choice.

6 6 Problem 6 To avoid aliasing (i.e. false spatial frequencies due to insufficient sampling density) an anti-aliasing filter can be used between the microscope objective and the CCD detector matrix used for recording digital images of the specimen. Let s assume that the detector elements in the CCD are arranged as shown in the figure below. 5 μm 10 μm y 4 μm 10 μm x The MTF curve of the microscope objective is given by: 1 MTF mm -1 where the spatial frequency (in units of mm -1 ) refers to the image plane of the objective, i.e. where the CCD is located. You have a choice of three different anti-aliasing filters, whose MTF curves are illustrated on next page.

7 7 MTF MTF MTF mm -1 mm -1 mm mm mm mm mm -1 Which anti-aliasing filter do you choose, and why? Can you see any disadvantages with using an antialiasing filter concerning the image quality? Explain! It is sufficient to treat the one-dimensional problem (for example x-direction only). Problem 7 It is a well-known fact that the maximum attainable signal-to-noise ratio (SNR) in light measurements depends on the number of photons detected during the measurement. Higher SNRs require more photons. The question is whether it is possible to obtain, in a given measurement situation, a higher SNR by using an image intensifier. An image intensifier is a device that produces multiple output photons for each incoming photon (this doesn t violate the principle of energy conservation, because we input electric power). Imagine that we couple an image intensifier, that produces 1000 secondary photons for each incoming one, in front of the detector. We then detect many more photons, but will we get a higher SNR? Answer this question by studying the SNR in terms of average signal level and standard deviation when performing repeated measurements. Assume that the detector output signal is directly proportional to the number of detected photons during the exposure time, and that both detector and image intensifier introduce negligible amounts of noise (i.e. only photon noise is present). (Formulas for standard deviation etc. can be found in App. 8 of Imaging Physics) Problem 8 Christmas is the proper time for making wishes. This year we shall wish for microscope optics that are useful for all wavelengths from X-rays right up to visible wavelengths. And while we re at it, let s wish for diffraction limited performance at all wavelengths. Supposing Santa delivers the goods, we will build two microscope set-ups for fluorescence microscopy. In both cases we will use soft X-rays (λ =.5 nm) for illuminating fluorescent specimens, which then emit light in the visible region (assume monochromatic light at λ = 550 nm). One set-up is according to Fig. 13 in the compendium Light Microscopy. In this set-up conventional (i.e. non-confocal) microscopy is used. A large specimen area is illuminated by an X-ray lamp, and the fluorescent light from the specimen is imaged by the objective and eyepiece. The other set-up is confocal, and according to Fig. 18 in the compendium. In this case we assume that the laser excitation light consists of X-rays. What is the highest spatial frequency (measured in the specimen plane) that could be imaged by these two systems? We will assume that the confocal detector aperture is very small. Problem 9 In cases where we have some prior knowledge concerning the specimen, it may be possible to extract useful information from images even if the sampling theorem has not been fulfilled. Consider the following case: We are using an NA = 0.90 microscope objective for imaging a periodic structure, whose spatial frequency we know is > 1.8 μm -1. The microscope used is non-confocal, and the imaging is incoherent at a wavelength of 550 nm. The image is digitally recorded with a sampling distance of 0.33 μm (measured in the specimen plane). In the recorded image we see a periodic, low-contrast pattern with a spatial frequency of 0.90 μm -1. Is it possible from these data to uniquely determine the spatial frequency of the periodic specimen structure, and if so what is the frequency?

8 8 Problem 10 When working with light microscopy at high magnification, the image quality is sometimes adversely affected by mechanical vibrations in the set-up. Let s assume that we are recording images with a (nonconfocal) microscope equipped with a high-quality 100/1.4 objective (i.e. 100x magnification and 1.4 numerical aperture). The specimen emits fluorescent light with a wavelength of approximately 580 nm. The effects of the vibrations can be described by a point spread function, psf vibrations, according to the figure below, where x is given in units of meters (in the specimen plane). We only consider a onedimensional case, with vibrations perpendicular to the optical axis of the microscope. psf vibrations 1 x x a) Determine MTF vibrations and PTF vibrations. b) Will the vibrations have any decisive influence on the image quality? (Motivation needed) Problem 11 In a non-confocal fluorescence microscope the specimen is uniformly illuminated with 436 nm light from a mercury lamp. The irradiance level is 1.0 mw/mm. The specimen is imaged using a 100x 4 objective, and the efficiency of the imaging process is such that it takes illumination photons to produce one fluorescent photon in the image plane of the objective. Images of the specimen are recorded by a CCD detector with 104x104 elements, each with a light-sensitive area of 10x10 μm. The detector is located in the image plane of the objective. The specimen is living and therefore moving. As a result, we want to record as many images per second as possible. We demand, however, that photon quantum noise must not reduce the signal-to-noise ratio below 0. The quantum efficiency of the detector is 50%. How many images per second can we record? Problem 1 We are imaging a specimen in reflected light (λ = 633 nm) using a confocal microscope with a very small detector aperture. As a result, imaging is purely coherent. The microscope is optimally focused on the reflecting surface. If, during scanning, the specimen vibrates in a direction parallel to the optical axis, this will cause intensity variations in the images. What is the maximum allowed vibrational amplitude if we cannot tolerate larger intensity variations than 5% due to vibrations. Scanning is performed with an N.A. = 0.65 dry objective. (Hint: If you encounter an equation you cannot solve analytically, use trial-and-error to get an approximate solution)

9 9 Problem 13 You want to record a specimen volume of 100μ m 100μm 100μm using confocal microscopy. The microscope optics are diffraction limited, and the detector aperture is so small that its influence on the resolution is negligible. You want to store the volume as a stack of digital images in a computer. Each pixel value in the images is represented by one byte of data. How many megabytes of data do you need to record if you don t want to lose any information due to the sampling process? The microscope objective has 100 magnification and a numerical aperture of 1.3. Oil with a refractive index of 1.5 is used as immersion medium. The specimen is fluorescenctly labelled, and you may use an average wavelength of 550 nm in the calculations. Problem 14 Single molecule detection has been demonstrated in fluorescence microscopy in recent years. Consider the following experimental situation: The fluorophore has an excited state lifetime of 3.5 ns (i.e. after excitation the molecule returns to the ground state emitting a fluorescent photon after, on average, 3.5 ns). The excitation intensity is high, which means that the molecule is excited again immediately after returning to the ground state. The fluorophore molecule is expected to last for ten thousand excitation/emission cycles before it is destroyed by the excitation light. Fluorescence photons are emitted isotropically in all directions. The fluorophore molecule is located in immersion oil (n = 1.5), and the objective is a planapo 40/1.0 oil immersion type. 50% of the photons that enter the microscope objective will be lost due to absorption/reflection in the optical system. The detector quantum efficiency is 40%. Estimate (i.e. reasonable approximations are allowed) the number of photons that can be recorded from a single fluorophore molecule before it is destroyed. How long will it take to record this number of photons? Problem 15 A fluorescence microscope according to Fig. 13 in the compendium Light Microscopy uses a highpressure mercury lamp with an emission spectrum according to the figure below. Int λ (nm) The specimen is labelled with a fluorophore whose excitation and emission spectra are given by the figure on next page. (The excitation spectrum shows how efficiently the fluorophore is excited by different wavelengths.)

10 10 Int. Excitation Emission λ (nm) You have a choice of 5 excitation filters that transmit the following wavelength bands in nanometers: 313 ± 5, 334 ± 5, 365 ± 5, 405 ± 5, 436 ± 5, 546 ± 5 Outside these wavelength bands the transmission is nil. Transmission spectra for available dichroic beamsplitters are given below. Light that is not transmitted is reflected. Note that these beamplitters are leaky, which means that there is a small transmission also for wavelengths that should be reflected. Transmission 5% leakage in reflection band d 1 d d 3 d 4 λ (nm) The transmission curves for the available barrier filters are shown below. 100% Transmission b 1 b b 3 b 4 λ (nm)

11 11 Select suitable excitation, barrier and dichroic filters for the given lamp and fluorophore. Remember that the excitation light is many orders of magnitude stronger than the fluorescence light. The microscope objective absorbs wavelengths below 350 nm. Include short motivations for the choice of filters. Problem 16 Technologicus microscopicus is an interesting little bacterium whose back is covered with green fluorescent stripes, see figure. The fluorescence intensity profile resembles a sinewave with a period length of 0.80 μm, and the contrast is high (nearly 100% modulation). Fluorescence intensity 0.80 μm x x You are recording a digital image of this stripe pattern using an ordinary (i.e. non-confocal) fluorescence microscope, equipped with a 5/0.65 dry objective (nearly diffraction-limited). In the image plane of the objective (see Fig. 1 in Light Microscopy compendium) a CCD area array sensor is located. The sensor comprises detector elements, each with an area of μm, see figure blow. The light sensitivity is uniform over the entire μm area of an element, and zero outside. y x 7.5 μm 7.5 μm a) Estimate what the modulation will be in the recorded image data from the CCD (you may use your own calculations and/or equations and figures in the compendium to get the result). b) Will the period length of the recorded pattern be correct (i.e. will aliasing occur or not)? (Motivation needed) Problem 17 The confocal principle can be used not only in microscopic imaging, but also in macroscopic applications. In this problem we will consider the use of a confocal set-up for imaging of the blood perfusion in the skin of a patient. After intravenous injection of a fluorescent substance, the skin fluorescence is recorded with an optical set-up according to the figure on next page. What optical

12 1 section thickness (measured as full-width-half-maximum) can we expect when using this kind of setup. The detector aperture is so small that it does not affect the depth resolution. ( λ laser λfluorescence 500 nm). Laser light Aperture Dichroic beamsplitter Detector Lens diameter 50 mm 0.50 m Air Fluorescence light Patient Problem 18 Aliasing, i.e. moiré-patterns due to insufficient sampling density, is often a problem when using area array sensors. In some cases, however, a bit of detective work can tell us whether a periodic pattern has been recorded correctly or not. In the present example we have no data to tell us whether the sampling criterion was fulfilled or not. An area array sensor with 56 x 56 detector elements was used, each with a light-sensitive area of 10 x 10 μm (see figure 1). The light sensitivity is uniform over the entire area of each detector element and zero outside, and the response is linear (i.e. pixel values are proportional to exposure). Using this sensor, a pattern according to fig. was obtained. Explain why we can be sure that in this particular case no aliasing has occurred, and that therefore the pattern in fig. is recorded with correct period length. (Hint: consider detector MTF) y x 10 μm Fig μm

13 13 55 (white) Pixel value 0 (black) Fig.. x Problem 19 You want to record, with as much detail as possible, a very large specimen volume (approx. mm x mm x mm) in a confocal fluorescence microscope. You are using a (dry) 5x objective with numerical aperture Suggest a suitable sampling density (sampling points/mm in the specimen volume) in all three dimensions. Would you expect that differences in resolution in the horizontal and vertical directions will be pronounced in this case? Explain! (The detector aperture is very small, and the objective is nearly diffraction-limited. Both excitation and fluorescence wavelengths are close to 550 nm.) Problem 0 In fluorescence measurements the light flux is often quite low, and therefore the signal-to-noise ratio (SNR) is often rather poor. The situation can be improved by prolonging the measurement time so that more photons are collected. One problem with using long measurement times is that the fluorescence intensity often decays gradually over time. The reason for this is that fluorophore molecules are destroyed by the excitation light (this is referred to as photo-bleaching). Consider the two cases shown in the figure below. In case 1 no photo-bleaching occurs. Therefore, the detection rate of photons (i.e. the number of photons detected per second) is I 0 = s -1 independent of the measurement time. In αt case photo-bleaching occurs, so that the detection rate is given by I() t = I 0 e, where I 0 = s -1 and α = 100 s -1. I 0 Case 1 Case a) Calculate for cases 1 and the maximum SNR that is theoretically possible as a function of measurement time τ. b) In case it is pointless to use very long measurement times. Where approximately would you say that this limit is (use a common sense estimation). t

14 14 Solutions. Problem 1 a) When closing the luminous field diaphragm, the illuminance level in the specimen remains constant. The illuminated area is, however, reduced. The total luminous flux is thus reduced, but this smaller flux is distributed over a correspondingly smaller area, leaving the illuminance level constant. b) Depth of field is controlled by the aperture diaphragm, which controls the total solid angle under which the objective collects light. Reducing this angle reduces the blur circle that occurs when an object is out of focus. Changing the luminous field diaphragm does not change this solid angle, and therefore does not affect the depth of field. c) Resolution is affected by the aperture diaphragm. It should be adjusted so that the numerical aperture of the condenser matches that of the objective, see Light Microscopy p. 19. d) If the eye pupil does not coincide with the exit pupil of the microscope, we will not be able to see the entire field of view simultaneously. This feels rather like looking through a keyhole. The reason for this phenomenon is that rays of all different directions (corresponding to different positions in the specimen plane) form a beam waist at the exit pupil. If the eye is held there all these rays can enter the eye simultaneously. If not, rays from some directions will miss the eye pupil. Problem λ If the phase angle ϕ changes by 1, corresponding to an optical path difference of, the light 30 intensity will change by approx. 0%. The optical path difference is then given by 550 ( n 1 ) d = 0.5d = nm d = 37 nm. Thus, we can expect that a glass thickness of 30 approximately 40 nm can be detected. Problem 3 True. Given the psf we can calculate, for an arbitrary object, what the image function will be. (The image function is the convolution of the object function and the psf, see Imaging Physics page ) True. This is a more limited case than the one above. Both are therefore true. False. For an arbitrary (i.e. not diffraction-limited) optical system we cannot deduce the psf from the Rayleigh limit alone. Systems with different psfs can have the same Rayleigh limit. Knowledge of the psf is necessary to completely characterize the imaging properties. True. If we know the Rayleigh limit of a diffraction-limited system we know what the psf looks like (Light Microscopy, p ). According to case 1 above, we then know exactly what the imaging properties are. Problem 4 a) If the Rayleigh criterion and optical section thickness are taken as resolution measures, we get: 0.45λ 0.45λ 8.5λ Volume resolution =. This means that the volume resolution is proportional to λ 3. N. A. N. A. 8πnsin ( α ) b) In order to solve this, we have to find out how the optical section thickness depends on the α 1 1 sin sin ( α) =.. 4 N A 4n Therefore the volume resolution is proportional to ( N. A. ) 4. numerical aperture. If we assume that N.A. is small, we get ( ) ( ) Problem 5 A suitable choice would be beam-splitter b and barrier filter 3. In this way most of the laser light will be reflected towards the specimen, and the fluorescent light will be efficiently transmitted by the beamsplitter (a would reflect very little laser light towards the specimen, whereas c and d would mean that we waste fluorescent light). Barrier filter 3 is the best choice, given the requirement that we must.

15 15 block all laser light (note that all beam-splitters transmit a few percent of the laser light reflected back from the specimen) and transmit as much as possible of the fluorescent light. Problem 6 With the CCD matrix used, we have a sampling frequency of 100 mm -1, meaning that the highest spatial frequency we can correctly record is 50 mm -1. The objective can reproduce much higher frequencies than this, and this is true also for the detector MTF, whose first zero value occurs at 00 mm -1 (x direction) and 50 mm -1 (y direction). Total MTF is the product the component MTFs, and therefore we need to add a component whose limiting frequency is 50 mm -1. We should therefore add the anti-aliasing filter with the lowest limiting frequency, which is 50 mm -1 ; the others cannot prevent aliasing completely. A disadvantage with using an anti-aliasing filter is that the modulation (i.e. contrast) for frequencies well below 50 mm -1 will also be considerably reduced. Therefore the image will not appear as sharp as it would otherwise be. Problem 7 SNR = mean value standard deviation = n k= 1 1 n i k k= 1 ( i i ) k n mean, where i k represents the value from an individual n 1 measurement. When using an image intensifier, all light values will be larger by a factor of F (= 1000 in this case). Thus, all i-values in the above equation will be replaced by Fi. We then get SNR = mean value standard deviation = n k= 1 1 n k= 1 Fi ( Fi Fi ) k n k mean, which gives exactly the same SNR as in the n 1 previous case. Using an image intensifier will produce both a larger output signal and higher noise, but the SNR will be the same (compare electron multiplication in a photomultiplier tube, which will produce the same effect). Problem 8 N.A. In the non-confocal case the highest spatial frequency that can be imaged is given by (see Fig. λ 8 in compendium Light Microscopy ), where λ is the fluorescence wavelength (all imaging is done in visible light - the X-rays are only used for flooding an extended area of the specimen with energy to excite the fluorophore molecules). In the case of confocal microscopy, however, the X-rays will be focused to a diffraction-limited (i.e. very small) spot by the objective. The same objective will then image fluorescent light from the (X-ray) illuminated spot onto the detector aperture. As a result, the N.A. N.A. spatial frequecy cut-off for the system will be given by + (see compendium Light λexc. λfluor. Microscopy p. 36). In the first case, the spatial frequency cut-off is given by N.A. 6 = N.A. m -1, which is equal to 3.6 N.A. μm -1. Thus, in this case resolution is not improved by using X-ray excitation. In the second case, the resolution is determined almost N.A. N.A. 8 entirely by the first term = = N.A. m -1, which is equal to 9 λexc N.A. μm -1. In this case we really get a benefit from the short wavelength of the X-rays.

16 16 Problem 9 Using an N.A objective, the highest spatial frequency that can be imaged is N. A = m 3.3 μm -1. Therefore, we know that the true spatial frequency is λ somewhere between 1.8 and 3.3 μm -1. The sampling frequency is 3.0 μm -1, meaning that the highest frequency that can be correctly recorded is 1.5 μm -1. We clearly have a case of aliasing, and frequency ν alias = 0.90 μm -1. Using the formula for aliasing in Appendix 8 of Imaging Physics, we get ν alias = n νsampling νreal, where n is a positive integer number. n = 1 is the only possibility, since = 9 n > 1 would give ν real > 5 μm -1. For n = 1 we get two possibilities, namely ν real =.1 and ν real = 3.9 μm is outside the possible range, and therefore the spatial frequency of the structure must be.1 μm -1. Problem 10 a) The OTF is the Fourier transform of the psf, normalized to unity at zero spatial frequency. Using the FT table on p. 310 in β, we find that OTF vibrations = πν e = e PTF vibrations = 0. 7 ν. This means that MTF vibrations = OTF vibrations, and b) With the objective used (asuming nearly diffraction-limited performance), we get an MTF objective that decreases almost linearly, reaching zero at N.A νlimit = = = m -1. We therefore get MTF λ 9 vibrations (ν limit ) = , and MTF vibrations (ν limit /) = This means that the vibrations will cause a serious deterioration in image quality. Problem An irradiance level of 1.0 mw/mm 15 means that = excitation photons reach hc λ the specimen per second and mm 4. Since the efficiency is only the number of photons will be reduced by this factor. Furthermore, because of the magnification, they will be spread out over an 7 area that is times larger. As a result, fluorescence photons reach the image plane per second and mm 4. With a detector element area of mm 3, photons will reach a detector element per second. Only half of the photons will be detected, and therefore photons will be detected per second. In order to get a SNR of 0, we need to detect (on average) 400 photons in a measurement. With the given photon flow, this will number is obtained in = seconds. Therefore, we can record a maximum of approximately three images per second.

17 17 Problem 1 sin(u / ) The light intensity as a function of defocus, z, is given by I(u) =, where u / 8π u = z sin ( α / ), and α is obtained from N.A. = sin α. A little trial-and-error qickly shows λ that u = 0.78 will give I(u) = From this we can calculate that 9 uλ z = = = m = 0.16 μm. This means that when the o 8π sin ( α / ) 8π sin (0.3 ) vibrational amplitude is greater than 0.16 μm, we will get a drop in light intensity that exceeds 5%. Problem 13 From page 39 in the compendium Light Microscopy we get the maximum spatial frequency components that can be imaged in all three dimensions: 4N.A νmax, xy = = = m -1 9 λ ( N.A. ) ( 1.3) 6 νmax, z = = =.0 10 m -1 9 λn According to the sampling theorem we must sample with the following frequencies to record all information: 7 νs, xy = νmax,xy = samples/m. 6 νs, z = νmax,z = samples/m. The total number of samples necessary in a 100μ m 100μm 100μm volume is ( ) = sampling points. With one byte of data per sampling point we get 1.4 gigabytes of data (which is no big deal today). Problem 14 4 We can expect that altogether photons will be emitted by the fluorophore molecule we are studying. How many of these can we expect to record? Since the photons are emitted isotropically we must find the solid angle under which we collect photons and compare this with the total solid angle of 4π. Spherical surface, area = A dφ φ α r Flat, circular surface, area = A N.A. = n sin α Specimen

18 18 A The microscope objective will collect photons under a solid angle of Ω =. We can calculate Ω r r dφ π r sin φ from dω = = π sin φ dφ, and integrate over φ from 0 to α. We then get r Ω = π( 1 cosα), i.e. the same result as eq. 10 in Light Microscopy. This is an exact formula. Since the task is to estimate the photon number, it is allowed to make a simpler small angle A π( r sin α) estimation of the solid angle which gives Ω = = π sin α. r r For an oil immersion objective with N.A. = 1, we get α = The exact formula then gives Ω = 1.55 steradians (the approximate formula gives 1.36) Thus, the fraction of photons that will be collected by the objective is = Because of 4π losses in the optics and detector, only the fraction = 0. 0 of these will be detected. Therefore, the total number of detected photons is expected to be =.5 10 (using the approximate solid angle, we get. 10 ). How long will it take to record this number of photons? On average, one photon will be emitted every 3.5 ns. This means that it takes = s = 35 μs for the molecule to emit ten thousand photons (of which we detect about 50), and then it will be dead. Problem 15 The objective transmits λ > 350 nm. For excitation we need λ < 400 nm. The only excitation wavelength that fulfills these requirements is 365nm. This wavelength is not ideal for excitation of the fluorophore ( 310 should be optimal), but the emission is intense and should be quite sufficient. The excitation filter should therefore be 365 ± 5. The dichroic filter should reflect 365 nm efficiently, but transmit as much as possible of the fluorescent light. d fulfills these requirements. The barrier filter must remove all of the remaining 365 nm radiation that is transmitted by the dichroic filter. This means that we have to choose b even though it will absorb some of the fluorescent light. Problem 16 a) Degree of modulation in recorded image = Degree of modulation in object MTF. We must determine the MTF-value for the entire imaging chain at the spatial frequency of the fluorescent pattern. MTF total = MTFoptics MTFdetector. MTF optics is given by the diffraction-limited curve in Fig. 8 in the compendium Light Microscopy. The spatial frequency of the pattern is ν = ( ) = m -1. The limiting frequency is N.A = = m -1 (we have assumed a wavelength of 550 nm). This 9 λ means that the spatial frequency of the pattern is 0.53 times the limiting frequency. From the curve in Fig. 8 in Light Microscopy we can measure that MTF optics at this frequency. For the detector we get (see compendium on Imaging Physics) ( πνl) MTF = sin detector, where ν is the spatial frequency of the magnified image of the πνl pattern, i.e. = m -1, and L is the width of a detector element, i.e. 5

19 m. Inserting these numbers, we get MTF detector = As a result, MTF total = Since the degree of modulation in the object was nearly 100%, we can expect that the degree of modulation in the recorded image will be nearly 30%. b) In order to correctly record the spatial frequency of the image we must have at least two sample points per period of the pattern (sampling theorem). The period length in the image 6 5 will be =.0 10 m = 0 μm. Since the distance between sample points is 7.5 μm, we get.7 sample points per period, and therefore the sampling theorem is fulfilled. Yes, the pattern will be recorded with correct period length. Problem 17 Equation 18 in the compendium Light Microscopy gives the FWHM optical sectioning thickness in o confocal fluorescence microscopy. In the current example α = arctan =.86. We get FWHM = =.7 10 m = 0.3 mm. This is far from microscopic resolution, but o 8π sin ( 1.43 ) can still give valuable information concerning skin structures. Problem 18 Let us denote the sensor width (10 μm) by D. The sensor then has a sampling frequency in the x 1 1 direction of. This means that aliasing occurs for frequencies above (= half the sampling D D frequency). A sensor with uniform sensitivity over the entire width D, and zero outside, will have an sin( πνd) 1 MTF given by MTF( ν ) =, where ν is the spatial frequency. At a frequency of we πνd D get an MTF-value of 0.64, and for higher frequencies we get lower values. This means that for spatial frequencies where aliasing occurs, we always get MTF < This in turn means that when aliasing occurs the degree of modulation of the pattern obtained will always be lower than 0.64 (the degree of modulation in the original pattern can never be higher than unity). A simple measurement in the figure gives a degree of modulation slightly above 0.8, and therefore we can conclude that aliasing cannot have taken place. Problem 19 Assuming perfect optics, and an infinitely small detector aperture, a confocal microscope can record 4N.A. ( ) N.A. spatial frequencies up to in the horizontal direction and in the vertical direction (p. λ λ 39 in compendium Light Microscopy ). With current data inserted into these equations, we get the frequencies 77 mm -1 (horizontal) and 18. mm -1 (vertical). In order to correctly record these maximum frequencies without aliasing, we have to use sampling densities of twice these values, i.e mm -1 and 36.4 mm -1. In reality we could probably use somewhat lower sampling densitites, because the MTF-value becomes very low as we approach the limiting frequency, see Fig. in Light Microscopy ). As we can see, there is a large difference in the maximum frequencies that can be recorded in the horizontal and vertical directions respectively (a factor of 40!). Therefore we can expect rather serious problems with different resolution in the horizontal and vertical directions.

20 0 Problem 0 The maximum SNR possible in a measurement where we can expect N photons to be detected is 4 N. In case 1 N = τ, where τ is the measurement time, and therefore we get τ αt I0 ατ 100τ SNR = 100 τ. In case we get N = I0e dt = ( 1 e ) = 100( 1 e ), and α 0 100τ therefore SNR = 10 1 e. In case one can easily calculate that at τ = 0.04 seconds we have reached 99% of maximum SNR, and certainly it is rather pointless to go on measuring after that.