Fluorescence Microscopy and Bioimage Processing

Question 1

What is the equation for magnification?

Answer 1

v/u = magnification
v = image distance (focal point to image)
u= object distance (focal point to object)

Question 2

How do you work out focal length?

Answer 2

lens maker formula

1/f = 1/v + 1/u

Question 3

How would you work out total magnification of an optical microscope?

Answer 3

Magnification Eyepiece
X
Magnification Objective
=
Total Magnification

Question 4

What is meant by Inverted Microscopy?

Answer 4

An inverted microscope is designed so that the object being viewed is upside down and the observer looks up into the microscope from below. This type of microscope is commonly used for observing samples that are suspended in liquid, such as in cell culture or live microorganisms.

Question 5

What is meant by upright Microscopy?

Answer 5

An upright microscope is a microscope that is designed so that the object being viewed is in an upright position, and the observer looks down into the microscope from above. This type of microscope is commonly used for observing samples that are on a slide, such as in biology and pathology.

Question 6

What is the role of an eye piece and whats its components?

Answer 6

Essentially a projection lens (5x to 15x
magnification)

Composed of Ocular peice and tube

Question 7

What is the role of a condenser in microscopy?

Answer 7

Condenser focus the light onto the specimen
* Aligns the light rays into a straight path
* Adjust for objective

Question 8

Explain Numerical Aperture in microscopy?

Answer 8

In microscopy, the numerical aperture (NA) of an objective lens is a measure of the cone angle of the light that can be captured by the lens. A higher NA means that the lens can capture light coming from a wider angle, which results in a higher resolution image.

The higher the resolution of the image, but also the greater the potential for aberrations and distortion.

Question 9

Explain Aberration and give 4 examples?

Answer 9

Aberration in microscopy refers to distortion or deviation of the image from the ideal image, caused by the imperfections in the optical system of the microscope. There are several types of aberrations that can occur in microscopy, including:

-spherical aberration, caused by the curvature of the lenses in the objective and eyepiece

-chromatic aberration, caused by the different wavelengths of light being refracted differently by the lenses

-field curvature, caused by the image being curved in the peripheral regions of the field of view

-distortion, caused by the image being stretched or compressed in certain areas

Question 10

How do we correct Aberrations?

Answer 10

-using achromatic lenses that are designed to reduce chromatic aberration or Fluorite or Plan apochromat

-using electronic correction, such as digital image processing to correct for distortion and other aberrations.

Question 11

Explain Refraction?

Answer 11

Refraction, or bending of light, occurs as light passes from one medium into another medium with a different refractive index.

Question 12

What is a refraction index?

Answer 12

Refractive Index: a dimensionless number that gives the indication of the light bending ability of that medium.

Question 13

What is the equation for Numerical Aperture ?

Answer 13

Numerical Aperture (NA) = n x (sin m)
m = angle of one-half the angular aperture (A)
dependant on the height of the objective from the tile as closer to the slide = larger angle
n = Refractive Index of imaging medium

The higher the total numerical aperture, the better the resolution.

Question 14

Define Resolution of a Microscope and give the equation?

Answer 14

The resolution of a microscope objective is defined as the smallest distance
between two points on a specimen that can still be distinguished as two separate
entities.

R = λ/(2 x NA) where λ is the wavelength and NA is the numerical aperture

Question 15

What is the principle of Fluorescence Microscopy?

Answer 15

The principle of fluorescence microscopy is based on the ability of certain molecules to absorb light at one wavelength and re-emit it at a longer wavelength, a process known as fluorescence.

The distance between the excitation and emission peaks is known as the Stokes shift

Question 16

Explain direct and indirect Fluorescence labelling strategies ?

Answer 16

Direct - Primary antibody is directly
conjugated to a fluorophore

Indirect - Primary antibody is indirectly detected by
a labelled secondary antibody

Question 17

What is the benifit of Fluorescence Microscopy?

Answer 17

Fluorescence microscopy gives you the advantage of better resolution.

Allows to collect images in more than one colour.

Question 18

What type of filters do Fluorescence microscopes use and why?

Answer 18

excitation filters: These filters are placed in front of the light source and are used to select the wavelength of light that will excite the fluorescent molecules in the sample.

Emission filters: These filters are placed in front of the detector and are used to select the wavelength of light that will be emitted by the fluorescent molecules in the sample.

Dichroic filters: These filters are used to separate the excitation and emission light paths. They are placed in the optical path between the excitation and emission filters, and are designed to reflect the excitation light and transmit the emitted light.

Question 19

What is the diffrence between epi-fluorescence microscope and Confocal Microscopy

Answer 19

Epi-fluorescence microscope collects light from the entire depth of the sample, resulting in a three-dimensional image projected onto a two-dimensional detector

while confocal microscope uses a pinhole aperture in front of the detector to collect light from a specific depth within the sample, resulting in high-resolution, two-dimensional images with minimal out-of-focus light and noise.

Question 20

What is a multiphoton microscope?

Answer 20

A multiphoton microscope is a type of fluorescence microscope that uses a non-linear optical process called multiphoton excitation to selectively excite fluorescent molecules in a sample. This process uses two or more photons of light to excite the molecules, providing high-resolution imaging of deep tissue samples without the need for mechanical scanning or exogenous markers, and it’s less phototoxic than single-photon excitation, making it ideal for live cell imaging.