PILM- Microscopy

what is mircoscopy

a technique used to view the smallest structures that aids in treatment, diagnosis and discovery of new disease or bodily mechanisms

what is light microscopy

Most common form that uses visible light to illuminate the specimen as it passes through it

• Images are magnified by a series of lenses that bend light to magnify images

• allows us to see morphology of tissue and cells

how to prepare sample for light microscopy

• Pick the proper specimen for what needs to be observed and prevent decay using a fixative (formalin)

• Dehydrate and embed in paraffin wax, slice thin using a microtome, and place on a slide

• stain slide using appropriate stain for what needs to be seen

• use glue with a similar refractive index to glass and add a coverslip to the slide ( makes image more clear)

how to use light microscope

• Place slide on stage, adjust light aperture to 0.5, and turn on the light, making sure it is at zero

• move stage to make sure tissue is in veiw

• adjust course, then fine focus (don’t adjust coarse on 40x)

• Adjust lighting as needed

strengths and limitations of light microscopy

strengths

• widely used technique

• high-resolution images

Limitations

• limited in the size and complexity of the sample that can be seen

what is fluorescent Microscopy

A microscope that relies of flourescent dyes/protein to label certain areas of a molecule/structure, causing them to emit light at certain wavelengths

• can allow us to track the movement of cells and molecules or tehri distribution

• used to provide insight into protein function and interaction with other molecules

how to prepare a sample for fluorescent microscopy

1. The sample must be fixed to preserve it using formaldehyde or methanol

2. The sample is made permeable to allow dye into the tissue using degradation solutions that break down the cell membrane

3. The sample is incubated in dye to allow it to bind ( too much dye can decrease contrast)

4. The sample in mounted on a slide and covered with a coverslip

how to use flourescent microscopy

• Select appropriate filters to isolate the wavelength of your dyes (enhances contrast and clarity); filters may differ between samples

• places slide in microscope and adjust brightness/contras to optimise photot quality ( overexposure can damage sample)

• - uses high quality camera for the best images

strengths and limitations of flourescent microscopy

• Allows for the visualisation of specific molecules and structures in cells

• Can be used to observe a cellular process

what is a polarising microscope

Uses polarised light to study the properties and materials, how they interact with light, and their structural characteristics

• light wavelengths vibrate differently on differnt plane

• passes light through a polariser, which only allows light that vibrates in a certian plane through

• all light used is vibrating on the same plane

what can be examined with a polarising microscope

minerals, crystals and tissues by examining how they interact with polarised light ( aka do they reflect it or rotate it or not)

• Helps identify types of materials, their composition, structure, and other properties

how to prepare a sample for a polarising microscope

1. Sample needs to be thin enough to see through but not transparent

2. mount on slide using Canada balsam or glycerine jelly as a medium

how to use a polarising microscope

1. Place the slide on stage, aligned with the polariser and adjust the focus and aperture settings (clearer image)

2. Rotate the sample to see how different angles reflect light differently and show different colours and pattern to get the best qaulity of images

what is a phase contrast microscope

Allows for the visualisation of otherwise invisible structures, cells and microorganisms

• creates contrast between different areas of a specimen by converting their refractive difference to difference in brightness

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what can you study with a phase contrast microscope

used to study morphology and behaviour of cell and microorganisms

• allows us to observe cell division and migration

• can tract immune cell movement in response to infection

• can study the effect of drugs on things like cancer

how to prepare sample for phase contrast microscope

sample must be thin enough for light to pass through, prepared using microtome steps and mounted with a coverslip using medium that matches coverslips reflective index (prevents distortion)

• can use tissues sample

• cells from culturing

• thicken samples need more prep via sectioning and staining

how to use phase contrast microscope

1. Place sample on the stage and check lens in free from debris

2. Adjust the light source and focus the lens for a clear, bright image and to create contrast

3. which is phase contrast settings adjust the phase ring until the desired contrast is reached

microscopy in cancer reaserch

Allows scientists to study and develop new treatments by watching how drugs interact with cells using phase-contrast microscopy

• Used to study angiogenesis or the development of tumours’ own blood supply

• Can be used to see structure in tumours to observe growth and cancerous cell featires to determine target of treatment

microscopy in infectious disease

allows us to observe pathogens + their interaction with host cells and bacteria and identify them to determine disease

• allows us to find most useful treatment and differentiate between different types of bacteria

• can investigate disease pathogenesis

• can be used to differentiate between gram + and - cells

microscopy in neruoscince

can study the structure and function of brain cells using confocal microscopy which makes 3D images of brain tissue and can study the connections of neurons

• A two-photon microscope can be used to study brain activity in real time in terms of sensory, learning and memory function

microscopy in cell biology

Used to study cell structure, features, function and movement using fluorescence microscopy

• allows us to observe cellular mechanisms in real time