“Anton Leeuwenhoek was Dutch
He sold pincushions, cloth, and such.
The waiting townsfolk fumed and fussed
As Anton’s dry goods gathered dust.
……………………………………………………………
Impossible! Most Dutchmen said.
This Anton’s crazy in the head.
We ought to ship him off to Spain.
He says he’s seen a housefly’s brain.
He says the water that we drink
is full of bugs. He’s mad, we think!
They called him dumkopf, which means dope.
That’s how we got the microscope.”
An excerpt from the children’s book “The Microscope” by Maxine Kumin which beautifully penned down how modern-day microscopy began with a simple lens of Leeuwenhock. The first step towards Robert Hooke’s compound microscope to the Nobel Prize-worthy phase contrast, super-resolution, and cryoelectron microscopy.
While Leeuwenhoek was observing everything with his simple (lens) microscope, his peer Robert Hooke used the first compound microscope in the mid-1600s. That 17th-century compound microscope is similar to the light microscope used in schools nowadays with a stage, light source, and three lenses. Hooke’s 1665 book Micrographia collated his microscopic investigations including the image of cell-like structure inside the piece of cork, which is the origin of the current use of the word “cell” in biology. Back then imaging and documenting involved painstaking intricate work, starting from preparing samples, focusing to sketching, with sometimes intriguing levels of imagination which was worsened by the optical aberrations in lenses. While working on diffraction gratings and different lenses, physicts Fritz Zernike realized the effects of phase in imaging and their applications to develop phase contrast microscopy to provide a better image. His invention of the phase-contrast microscope earned him the Physics Nobel prize in 1953.
The greatest challenges in microscopy have been inherently low contrast of biological samples and the fundamental limit to the resolving power of the microscope. A number of different methods for increasing contrast have been tested and established including imaging phase, usage of lower wavelength light and utilization of various stains and fluorescent dyes. Carmine staining used for human brain sample staining is one of the earliest documented report from 1858, which was later followed by Golgi’s silver staining and Gram staining to name a few. Combination of staining and fluorescence detection led to the development of fluorescein as the first fluorescent stain in 1871. Almost a century later, green fluorescent protein (GFP) was first isolated from the jellyfish Aequorea Victoria and was later shown to be successfully expressed and fluoresced outside jelly fish by incorporating in the β-tubulin gene. The brilliant discovery and development of GFP by Osamu Shimomura, Martin Chalfie and Roger Tsien revolutionized the world of microscopy, and their contribution to science was recognized with the Nobel Prize in Chemistry in the year 2008.
The field of florescence microscopy has been further advanced by the development of variation of mutant GFP (blue, cyan and yellow florescent protein) and isolation of other fluorescent proteins like dsRed from coral species Anthozoa. A major breakthrough with the discovery of fluorescent proteins was devising methods to fuse the genes (protein) of interest with the genes for fluorescent protein and express it in a cell or tissue leaving it relatively unperturbed. Importantly, fluorescently labeled antibodies formed the basis of immunofluorescence assays which opened up the field of antibody labeling to study protein expression and function in cells and tissues. To aid microscopy, various tissue clearing methods like Clarity have been recently developed which removes the lipids from the tissue, making it as clear as glass without disturbing the tissue structure and maintaining protein stability for antigen-antibody interaction for labeling.
The early transmission fluorescence microscope led to the invention of epi-fluorescence microscopes. Several innovative illumination modes have been developed since then which include confocal, multi-photon and light-sheet microscopy. In confocal microscopy, a pinhole is utilized such that only in-focus light is collected which drastically reduces the out of focus background and provides a clear and crisp highly resolved image. In addition, confocal microscopes are capable of optical sectioning, three-dimensional reconstruction and obtaining better fluorescence contrast by fluorophore excitation using lasers. Utilization of high energy lasers comes with a price of photobleaching due to longer exposures which lead to newer techniques such as multiphoton microscopy. The most common form being two-photon microscopy wherein two photons with half the energy can excite emission of one photon. Thus, it can penetrate deeper in tissue sample with lower energy and reducing the damage due to imaging.
For over a century, the resolution of microscopes was limited as only the bright light was used conventionally. Utilization of lower wavelength source like electron beams rather than photons from bright light increased spatial resolution and the strength ofelectron microcopy (EM) was realized. To study the fine cellular structures and protein complexes at molecular resolution, cryogenic EM was developed. CryoEM involves imaging of frozen-hydrated specimens at extreme temperatures at which molecules are almost stationary. For developing this astonishing imaging technique, Jacques Dubochet, Joachim Frank and Richard Henderson were jointly awarded 2017 Nobel Prize in Chemistry. Moving forward and breaking its own limits, Cryo-EM has reached atomic resolution levels recently. To circumvent the difficulties of EM, super resolution mode of light microscopy was developed by breaking the diffraction barrier of light used for imaging. This discovery earned 2014 Nobel Prize in Chemistry for Stefan Hell, William Moerner and Eric Betzig.
The discovery of spectrally distinct fluorescent proteins allowed multichannel (multi-colour) fluorescence imaging and opened up ways to study the interaction between different fluorescently labeled proteins. The problem of signal bleeding from one fluorescent channel to its neighbor was realized while expanding the number of fluorophores that can be simultaneously imaged on a microscope. This led to the innovative labeling of proteins with rare earth metals probe and development of multiplexed ion beam imaging by time of flight (MIBI-TOF). It uses bright ion sources and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies at subcellular resolution.
As the limit of imaging speed and the level of detail that can be imaged even inside live animals are setting new records, it also brings in new challenges for the computing technology used to store and analyze the data being generated. Further, to make the visualization of miniscule world more affordable for not so privileged young minds and scientists, we need the cheaper versions which will break down the price barrier and cater to the curiosity and excitement of scientific exploration. We need to develop more devices like Manu Prakash’s foldscope or the paper microscopes.
