Microscopy- simple, fluorescent, and electron

Simple microscope

Uses- view objects or specimens in a clear and magnified way.
The main component present in the microscope you use in your lab- double convex lens (biconvex lens) with a short focal length.
Biconvex lenses can focus parallel light rays into a single point, which is important for visualizing intricate details on your specimen. The double lens also helps in providing good magnification of almost up to 300X
A shorter focal length corresponds to a higher magnification.
The sample should be stained.
The image produced by a microscope is erect, magnified, and virtual (cannot be projected on a screen)
Parts of the microscope:
- Eyepiece- 10X or 15X, the main lens
- Base- for mechanical support
- Tube- connects the eyepiece to the objective lens
- Objective lenses- 10X, 40X, 100X
- Nosepiece- the objective lenses rest on this- can be rotated
- Diaphragm- control the amount of light
- Stage- platform used for placing the slide
- Stage clip- used to hold the slide on the stage
- Knobs- two types- coarse adjustment and fine adjustment
- Condenser- focus the light on the sample
Magnifying power
- M= 1 + D/F
  - M magnifying power/ magnification
  - D least distance of distinct vision
  - F focal length
Benefits-
- Ease of use
- Used for most microbiological applications
Limitations-
- Low resolution
- Low magnification compared to compound microscope
- Staining required

Used for seeing transparent or translucent specimens without staining them
Basic principle- converts phase shifts in the light to brightness shifts in the specimen.
The microscope alters the wavelength of light passing through the specimen and generates an image.
As you have learned in your 12th-grade physics, when light travels from one medium to another, the velocity changes. This deviation is proportional to the difference in refractive index between the two mediums.
Benefits-

Quick and efficient
- No fixing or staining is required
- It enhances the contrast of transparent images
- Biological processes can be seen and recorded as we are not killing or staining them
- Best for real-time monitoring
Limitations-
- The boundaries cannot be visualized properly
- Ideal for thinner samples only
- Expensive instrumentation
- More light is needed for phase contrast, hence more power consumption

The use of florescent light to visualize structures of organic and inorganic substances
A high-intensity light source excites the florescent components (fluorophores) present in your sample. When the sample emits a lower energy light of a longer wavelength, the objective lens of the fluorescence microscope detects the image.
The main components are two filters-
- Excitation filter- transmits only light that excites the specimen dye
- Emission filter- filters out the light at the excitation wavelength after it has interacted with the sample
How does fluorescence work? When certain compounds in your specimen are hit by a photon, they can absorb the energy of that photon to get into an excited state. This is filtered out by your emission filter and finally detected by the objective lens.
Benefits
- To study specific features of your specimen like your microorganisms
- It can enhance the 3D features of the sample
- Helps us visualize specific proteins (by tagging them with florescent molecules)
- Helps us see the motility and ion transport processes
- Medically, they help detect tumor cells and other anatomical features
- Highly sensitive and selective
Types-
- Epi-Florescence microscope
- Confocal microscope
Limitations-
- The fluorophores lose their fluorescing capacity in a process called auto-bleaching
- Cells are prone to phototoxicity

A beam of electrons instead of a beam of photons (light)
It can be used to get high-resolution images of biological and non-biological substances.
Working principle- A beam of electrons passes through a vacuum tube and eventually reaches the sample. A high voltage is required for this. The beam of electrons is focused on the sample with the help of magnetic lenses
An electron gun releases the beam of electrons into the vacuum tube.
The interaction between the electrons and the sample generates an image.
Two main types- Transmission Electron Microscopy and Scanning Electron Microscopy
Applications-
- Identify the structure of cells, tissues, and organelles
- Diagnosis of viral infection
- Identify cell culture isolates
- Select PCR primers
- Quality control
- Identification of 3D structures of certain proteins which cannot be detected by X-ray crystallography or NMR spectroscopy
Limitations
- Expensive
- Not available in many laboratories
- Requires a special technique- fixation
- Cannot detect low levels of virus load

TEM

It is used for viewing thin samples
The specimen should be ultra-thin
It is used to study the structure and morphology of cells and tissues
Gives 2-D projections of the sample
Its magnification power is almost 2 million times better than a regular light microscope!
Main components-
- Electron gun – to produce the beam of electrons
- Image producing system- with an objective lens and intermediate & projector lenses
- Image recording system- consists of a florescent screen with a digital camera attached
The image obtained will be monochromatic (black and white)
It can also be captured digitally and stored on a computer, where we can add colors to make the visualization better
Limitations-
- It is not easy to handle a big instrument
- Sample preparation is tedious
- Expensive equipment

SEM

It is used to visualize the surface of our specimen
It gives the 3-D projections of the surface of the specimen
The electrons are reflected or knocked off the surface of the cell or specimen
Benefits-
- No specific sample preparation techniques are required
- Can accommodate large bulky specimens
- Higher resolution than TEM
Limitations-
- Special training required for equipment handling
- A small risk of radiation exposure
- Vacuum environment