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Homework has two parts, detailed description follows:
Important equations
$$ Z = \frac{\Delta}{f_{ob}} \frac{d}{f_{oc}} $$
Let us consider a thin convex lens (spojná čočka) with focal point distance of 10 cm. Find the magnification for a red object of height 6 cm, placed in the distance of 12 cm in front of the imaging plane.
Solution Given: $f=0.1~m, y=0.06~m, a=0.12~m$ Magnification is the ratio of the size of the displayed object to the size of the original object $Z = \frac{y'}{y} = \frac{a'}{a}$ and the imaging equation gives us $a' = \frac{f \cdot a}{a - f}$ and finally, after substituting $a'$, we get $$Z = \frac{f}{a - f} = \frac{0.1}{0.12 - 0.1} = 5$$
Consider a simple microscope formed by two thin convex lenses with objective focal length 5 mm and diameter of 1 cm, tube optical length 100 mm and eyepiece focal distance of 25 mm. Draw a schematic picture of the microscope and compute its magnification.
Solution Given is $f_\text{ob} = 0.005$, $f_\text{oc} = 0.025$, tube length $\Delta = 0.1 m$ and conventional optical distance $d=0.25 $m. By simple insertion into the magnification equation, we get
$$ Z = \frac{\Delta}{f_{\text{ob}}} \cdot \frac{d}{f_{\text{oc}}} = \frac{0.1}{0.25} \frac{0.005}{0.025} = 200. $$
Consider a simple microscope with an objective magnification of 40, eyepiece magnification of 10 and tube length of 12 cm. Compute the magnification as well as the focal lengths of the objective and the eyepiece.
Let us consider a microscope with eyepiece numerical aperture (NA) of 0.4, using illumination by light of wavelength 650 nm. Compute the maximal resolution (minimal distance between distinguishable points) we can achieve in this setting.
Solution From Abbe-Rayleigh criterion: $$ d = 1.22 \frac{\lambda}{2 NA} = 1.22 \cdot \frac{6.5 \cdot 10^{-7}}{2 \cdot 0.4} = 990 ~n \text{m} = 1 ~\mu \text{m} \, $$
Let us consider a lens $L$ with a half-cone angle $\theta=30^\circ$ in immersion oil ($n = 1.33$) and normal light illumination ($\lambda$ = 650 nm). Which light (wavelength and color) do we need to use to get the same resolution capabilities when using air ($n=1$) as a diffraction medium?
Histopathology images are microscopy images of small tissue samples (cuts or slices). Since cells are usually colorless, we use the technique of staining to make important structures visible in the microscope. For this, the tissue probe is put into a solution with two dyes, most commonly Hematoxylin and Eosin. They are both colorless chemical compounds with different properties
Different dye concentration leads to differently colored images. This effect can be further emphasized by different lighting conditions during image acquisition. To apply image segmentation or classification techniques, we want the color variability to be minimal. To achieve this, we usually apply a stain normalization technique.
Color (de-convolution) According to the Beer-Lambert law, there is an exponential relationship between the intensity of the transmitted light $I_\lambda(\mathbf{x})$ at wavelength $\lambda$ in a 2D point $\mathbf{x}$ and the amount of absorbed dye $n(\mathbf{x})$, given by the following equation
$$ I_\lambda(x) = I^0_\lambda(x) {\rm e}^{-m_\lambda n(x)}$$
where $I^0_\lambda(x)$ is the intensity of the incident light and $m_\lambda$ is the attenuation coefficient of the stain. Let us define the optical density $d_\lambda(\mathbf{x}) = \log( \frac{I^0_\lambda(\mathbf{x})}{I_\lambda(\mathbf{x})})$, leading to $d_\lambda=m_\lambda n(\mathbf{x})$. We now assemble the individual $d_\lambda$ values into a 3D vector, corresponding to standard RGB colors:
$$ \mathbf{d}=(d_R,d_G,d_B)=M \mathbf{n}(\mathbf{x}) $$
where $M$ is a so-called color mixing or color convolution matrix. The vector $\mathbf{n}(x)$ also has three components - two representing the hematoxylin and eosin concentrations and the third representing the residual attenuation - impurities and model imperfections. The remaining step is to concatenate vectors $\mathbf{d}(\mathbf{x})$ and $\mathbf{n}(\mathbf{x})$ from all pixels in the image into $3 \times P$ matrices $\mathbf{D}$ and $\mathbf{N}$, where $P$ is the number of pixels.
$$\mathbf{D} = \mathbf{M} \mathbf{N}$$
The key tasks are the estimation of the mixing matrix $\mathbf{M}$ and reconstruction of the stain density image $\mathbf{N}$ from the observed optical density image $\mathbf{D}$ and thus identifying the concentration of the dyes. This is called color or stain deconvolution. Then it is possible to create images with an arbitrary but identical appearance by applying the same color mixing matrix $\mathbf{M}$ to all images. The resulting images are called stain normalized.
Processing hints
We start with an image $I$, where each pixel $I(x) = (r, g, b)$ has the color information in RGB color space. We then compute the optical density image $D = -\log{\frac{I+1}{I_0}}$ with the maximal intensity $I_0 = 255$ in our case (the +1 is to avoid zero within the logarithm). With a given color matrix $M_A$ we get the stains $N(x)$ by solving $D(x) = M_A \cdot N(x)$ in each pixel $x$. Note: To multiply all pixels with a $3 \times 3$ matrix at once, the (rgb) image ( $i \times j \times 3$ ) must be reshaped to shape ($i \cdot j \times 3$) and/or even transposed.
Homework
Residual
Hematoxylin
Eosin