As readers of my blog will be aware, I enjoy imaging natural things. For me ” Natural World” includes the very big (like galaxies) and the really rather small (like cells). I’ve decided to include some Microscopy (or Photomicrography) in my blog, so by way of a start I thought I’d include some images of chromosomes, the data bookshelves of life on our planet.
Chromosomes require fairly substantial magnification and the simplest way to achieve acceptable resolution at x1000 with a standard optical microscope is to use oil immersion microscopy; this is how the following images were taken …
Chromosomes during Mitosis
Mitosis is the process of duplicating & splitting the nucleus / genetic material of a eukaryotic cell. It is normally followed by cytokinesis which completes cell division, resulting in 2 identical daughter cells. Mitosis is used for growth & asexual reproduction, it is distinct from meiosis (used for sexual reproduction) and binary fission of prokaryotic cells. Other than the ‘normal’ interphase state of cells, Mitosis has 4 main phases & these allows us to view & photograph chromosomes:
- Prophase – During interphase the genetic material will have been duplicated, now the nucleus swells and the chromatin within, coils up to form chromosomes; each consisting of two chromatids. Simultaneously the centrosome duplicates & each resulting diplosome moves towards opposite ends of the nucleus.
- Metaphase – In most cases the nucleus has now become indistinct. The chromosomes line up across the centre of the cell and protein spindle fibres develop across the cell, coordinated by the centrosome at either side of the cell.
- Anaphase – Each chromatid pair splits, forming sister chromosomes. The protein spindle threads now pull the chromosome sisters apart, to opposite ends of the cell.
- Telophase – Each group of chromosomes are now at either end of the cell, they are now enveloped by a new nuclear envelope. The chromosomes now uncoil back in to chromatin and are no longer distinct for us to see.
To complete cell division the cells cytoplasm will now cleave and two new sister cells will form; this is cytokinesis.
So what of it? Well the chromosomes are particularly distinct during Metaphase & Anaphase and that gives us a great opportunity to photograph them. The process doesn’t take very long, Metaphase can be over in 15 minutes; so we need to look at a whole bunch of cells & pick out the few that have been ‘caught in the act’ as it were. The micrograph below is of onion cells dividing mitotically near the root tip …
The image was taken at 1000 times magnification (x10 ocular, x100 objective) and used oil immersion to improve the image (see below). The cell in the centre of the picture displays distinct blue stained chromosomes and just possible to make out is the occasional spindle fibre. Two other cells can be seen at different stages.
Giant Chromosomes in Fruit Flies
Drosophilia, the favourite of geneticists across the ages are one of a few insects that have an unusual characteristic. They develop ‘giant’ or polytene chromosomes in their salivary glands. DNA is replicated many times, without cell division taking place. This can lead to large banded chromosomes with perhaps 1000 times the normal compliment of DNA, 85% of which may be concentrated in to the dark bands. These give us another excellent opportunity to photograph chromosomes.
Image details: Magnification x1000, Light Microscopy with Oil Immersion, Canon 300D ISO 100 TV 1.3s, 4 image stack using CombineZM
Technique & Equipment
How were these images taken? The basic equipment is pictured below:
Referring to the image above, from bottom to top: Light Source, Slide, Oil, Objective Lens, Ocular lens, photo adapter, DSLR
ie. Immersion Oil is carefully placed here (picture below), between objective lens & slide coverslip.
The microscope is carefully focused away from the slide until best focus is found. One draw back of this method is that the focal plane is absolutely tiny. The advantages are simple, at high magnifications light microscopes loose resolution due to refraction at the glass air boundaries; by using immersion oil with the same refractive index as the glass, 2 such boundaries are removed & resolution is restored.
I shall add more microscopy images in time to come.