2.1.1 (a) Microscopes

Learners should be able to demonstrate and apply their knowledge and understanding of:

HSW1 Use theories, models and ideas to develop scientific explanations.

HSW7 Know that scientific knowledge and understanding develops over time.

What should you know from GCSE and KS3?

How to use a light microscope
the parts of a eukaryotic cell (GCSE level).
Light travels in straight lines and reflects off objects in order for us to see them.
Lightwaves have shorter and longer wavelengths.
How the human eye can see objects.
What is a focal point, how do lenses refract light into a focal point?

Light microscopes

Laser scanning microscopes (aka confocal microscopes).

How do laser scanning confocal microsopes work?

EXTN - Medical uses for confocal microscopy - VIrtual Biopsies!

More extn work - read the background behind the 2014 Nobel prize for Chemistry - for super resolved fluroescence microscopy. This has allowed scientists to use fluorescence microscopy to see individual molecules in living cells.

Electron Microscopy

Transmission electron microscopy (TEM)

Scanning electron microscopy (SEM)

How are samples prepared for electron microscopy?

Disadvantages of electron microscopy

EXTN - Atomic force microscopy

Past Paper questions and markschemes

This video goes from light microscopes, to electron microsopes and further than the spec into scanning probe microscopy (SPM). The textbook has extension work on Atomic force microscopy on page 23 which I believe is a version of SPM.

This video is from HHMI and can be used alongside the activities here https://www.biointeractive.org/classroom-resources/what-van-leeuwenhoek-saw

How do we see cells?

Light microscopes

The original microscopes used light and lenses to magnify whatever was on the stage. The light microscopes we use at school work the same way.

On a compound light microscope, has a thin specimen or slice of a specimen placed on a glass slide.

Light from a light source underneath the sample goes up through the slide and into an objective lens. This is called brightfield microscopy.

If you are looking at an opaque object, the light source will need to come from above the specimen.

If an entire specimen is illuminated at once, it is called widefield microscopy.

Most microscopes have more than one objective lens - each with a different magnification - to allow the researcher to look at the sample with different magnifications.

The compound light microscope has 2 lenses - the objective and the eyepiece lens.

The objective lens is closest to the specimen slide. This is the lens that produces a magnified image. This image is magnified again by the eyepiece lens.

Early microscopes had trouble with chromatic aberration but having the combination of objective and eyepiece lenses removed this interferrance from what scientists could see. (An example of an image with chromatic aberration is the picture of the wolf on the right whilst the image of the wolf on the left has a filter to remove the aberration)

Light Microscopes - use wavelengths of between 400 - 700nm. You cannot distinguish between structures closer than 200nm using a light microscope. They will appear as one object. But the light microscope cannot produce higher magnification without losing much resolution.

Chloroplasts are visible within cells of the forest-growing moss Plagiomnium affine. www.the-scientist.com

Small Muscular Artery www.medcell.org

Laser scanning microscopes (aka confocal microscopes).

Laser scanning confocal microscopes use laser light (light at a high intensity) to take many photos at different layers of the sample. The unfocused light from other planes is blocked out. If you take many in focus photos and combine them together, you are able to get a 3d image.

Specimens are stained - sometimes with florescence - so you can identify where particular organelles are located.

How do laser scanning confocal microsopes work?

Specimen is treated with a fluorescent chemical. Fluorescent chemicals can absorb the 'excitation' light rays and radiate them back.
The specimen is placed.
The laser hits a mirror which reflects it onto the specimen. This mirror reflects laser light only but the other light wavelengths are able to pas through it.
A single spot of focused light (point) is moved or scanned across a specimen. This is called point illumination. It is moved in one focal plane.
This point of light excites the fluorescent dyes present and they emit light. The emitted light from the desired focal plane passes through a pinhole aperture. These are the focused rays. These are the rays that are picked up by the detector.
Any rays being emitted from areas of the speciman not on the focal plane will not pass through the pinhole aperture. These would be the unfocused rays, but because they do not pass through the pinhole they are not detected by the detector. This increases that resolution of the image as the blurry out of focus rays are not picked up.

Why is is called confocal? There are two pinholes, one at the laser and one at the detector. These pinholes force both the light from the laser and the light being radiated from the sample follow the same path. They are on the same focal plane => confocal. Con from the latin for 'with or together'.

2D images - The spot illuminating the specimen is moved across one focal plane only and a high resolution image is produced.

3D images - The spot illuminating the specimen is moved across different focal planes and these high resolution images can be stacked to create a 3D image.

EXTN - Medical uses for confocal microscopy - VIrtual Biopsies!

Li, J., Garfinkel, J., Zhang, X. et al. Biopsy-free in vivo virtual histology of skin using deep learning. Light Sci Appl 10, 233 (2021). https://doi.org/10.1038/s41377-021-00674-8 www.nature.com/articles/s41377-021-00674-8#Sec1

(a) Standard tissue biopsy, followed by tissue fixation, processing, and staining results in microscopy slides for pathological interpretation. (b) By employing the trained deep neural network that takes a stack of RCM images of unstained intact skin as input and instantly generates corresponding virtually stained tissue images, the reported deep learning-based virtual histology of skin may provide a unique avenue to biopsy-free, label-free clinical dermatological diagnosis. Each time, a stack of seven axially adjacent RCM images is fed into a trained deep neural network VSAA and transformed into an acetic acid virtually stained tissue image that is corresponding to the central image of the input stack, so that a stack of N images can be used to generate N-6 virtually stained 3D output images that are axially adjacent. Following this acetic acid virtual staining, a pseudo-H&E virtual staining step is further performed by a trained deep neural network (VSHE).

Electron Microscopy

Light microscopes can produce images of higher and higher magnification but these images have reduced resolution meaning the images are blurry. Electron microscopes have been developed to provide highly magnified images of a much higher resolution. They use a beam of fast travelling electrons with a wavelength of 0.004 nm compared to the lightwaves wavelength of 400 - 700nm. Electron microscopes are able to produce images with magnifcations of x500000 but with also with a resolution high enough to see the components of cellular ultrastructure.

How does an electron microscope work?

Electrons come out the heated filament.
They are focused onto the specimen using electromagnets.
The electrons either pass through the specimen (Transmission) or reflected off the specimen (Scanning) and the resulting patterns are detected and processed into images by a computer.

Transmission electron microscopy (TEM)

Beam of electrons transmitted through a specimen and the electrons lose energy if they pass through certain regions of the specimen. The electrons and their energy levels are detected and this information is processed into a black and white 2D image (they are sometimes coloured after they are produced eg photoshop)
TEM has a resolution of 0.5 nm. The best we currently have.
Process takes place in a vacuum.
The specimen is chemically fixed (dehydrated and stained with metal salts) so specimens are dead.
can magnify up to 2 million times.

https://histologyslides.med.umich.edu/Histology/EMsmallCharts/3%20Image%20Scope%20finals/128%20-%20Monocyte_001.htm

Scanning electron microscopy (SEM)

Beam of electrons are fired across the surface of a whole specimen and electrons from the specimens surface are reflected (secondary electrons) and are focused on a screen.
Specimen can be whole but is viewed in a vacuum so is dead.
Specimens are also coated in metal salt stains.
Image is 3D and black and white (can be coloured using computers).
Magnification of x15 to x200 000.
Resolving power (the resolution) is from 3 - 10 nm.

Also a monocyte

How are samples prepared for electron microscopy?

Electrons in electron beams are smaller than atoms so the channel they are passing through cannot have any molecules in the way. For this reason electron microscopy needs to take place in a vacuum.

Samples will be frozen or 'fixed' with chemicals. Samples can be stained with heavy metals and dehydrated. This is why we cannot look at living samples in the electron microscope - they just wouldn't survive the preparation process.

TEM samples may be put in resin.

SEM samples may be fractured to expose the inside and then covered in heavy metals.

https://www.leica-microsystems.com/science-lab/life-science/brief-introduction-to-freeze-fracture-and-etching/

Disadvantages of electron microscopy

Extremely expensive.
Need a controlled and dedicated environment.
Specimens can be damaged by the electron beam and the preparation process.
Artefacts (structures that are produced due to the prepararation process) may be mistaken for actual structures in the cell.
Need highly trained technicians to run the microscope.