hey everybody it's mr luther i'm back with you for section 2.3 of openstax microbiology instruments of microscopy 2.1 we talked about the properties of light in section 2.2 talked about the history of the microscope here today again i hope that you will be able to identify and describe the parts of a bright field microscope hope that you will be able to calculate total magnification for a compound microscope and hope that you will be able to describe the distinguishing features and typical uses for various types of light microscopes electron microscopes and scanning probe microscopes here we go all right so early pioneers of microscopy opened a window into the invisible world we talked about the fact that anton van leeuwenhoek first to really discover the microbial world with a very simple microscope but there were other microscopes at the time and other people working on this continuing in 1830 joseph jackson lister created an essentially modern light microscope and the 20th century saw the development of several microscopes that leveraged non-visible light you know we're talking you know the previous microscopes early microscopes only were able to use visible light now we can use things such as ultraviolet light these advances led to major improvements in magnification resolution and contrast many types of microscopes fall under the category of light microscopes we will talk about bright field microscopes dark field microscopes phase contrast microscopes differential interference contrast microscopes fluorescent microscopes confocal scanning laser microscopes sounds like something from austin powers and dr evo i weren't kind of scanning laser microscopes on my sharks thank you very much two photon microscopes are the last one that we will talk about all right starting with bright field microscopes uh thing you need to understand just like the name implies it produces a bright image you can use stains to increase the contrast when you look at the parts of a light microscope here is how it all works together you start off with the eyepiece or the ocular lens that has a power of 10x it magnifies things 10 times all by itself then you follow the body tube down you get to the rotating nose piece which contains the objective lenses the objective lenses have various powers from 4x to 100x and it works together to get a total magnification and you simply multiply the ocular times the objective if i'm using a 10x ocular and a 4x objective my total magnification would be 40x if i'm using a 10x of ocular and a 100x objective now my total magnification is going to be one thousand ten times one hundred the other being viewed is called the specimen the specimen is placed on a glass side which is then clipped into place on the stage right here this flat piece is the stage it holds the slide once the slide is secured the specimen on the slide is positioned over the light using the x y mechanical stage knobs you work in the x y axis that is left to right back and forth that's all the stage knobs can do is stay in that plane and move the specimen around they do not raise or lower the stage those are other controls on the microscope once the specimen is centered over the light the stage position can be raised or lowered to focus the image you raise or lower the stage using the coarse focusing knob and then you will find focus using the fine focusing knob which is number five four is course five typically how you do this you put your get your slide get it centered and then you take the course and you lower the stage to its lowest position and then you slowly turn the course focus so the stage raises until you get an image then you use the fine focus to tweak it and get a nice clear image you do that to avoid breaking the slides the illuminator provides intense lighting technically typically excuse me due to a high intensity bulb below the stage right there is the illuminator light from the illuminator passes up through the condenser lens which focuses the light into the objective so you get maximum maximum illumination if less than maximal light levels are needed the amount of light striking the specimen can easily be adjusted opening or closing the diaphragm the diaphragm is under the stage and it controls how much light actually enters you can open it for max light or you can close it or sometimes they have smaller openings so you can control the amount of light that comes in in some microscopes you also may have a rheostat which allows you to dim or brighten the actual light source itself in a bright field microscope different colors can behave differently as they interact with chromophores pigments that absorb and reflect particular wavelengths of light in parts of a specimen chromophores are artificially added to the specimen using stains which serve to increase the contrast and resolution here are some bright field images notice the bright background there are potentially stains that have been used here to enhance the color increase the resolution and the contrast but again you see a bright background with a lit up image another thing you can do with the bright field microscope if you turn to the 100x objective you must use immersion oil or oil immersion or oil of immersion always to say this and very high magnifications resolution may be compromised when light passes through the small amount of air between the specimen and the lens this is due to the large difference between the refractive indices of air and glass the air scatters the light rays before they can be focused by the lens so all you need to do to combat that is to add a drop of immersion oil with the 100x lens right here you'll see the drop of oil it connects the tip to the glass of the slide and all that does a lot actually the refractive index of oil and glass are virtually the same so the light coming up from the light source is able to be focused into the objective lens instead of the scattering effect that you see without the oil this will increase the resolution of the image just to show you the type of resolution you can you know here you will see an image with uh the high objective the 100x lens but it's not very good when you add immersion oil you greatly increase the contrast in the resolution because now you're focusing all the light it's being refracted into the objective lens dark field microscopes a dark field microscope is a bright field microscope that has a small but significant modification that should be modification i'm sorry about that to the condenser a small opaque disc about one centimeter in diameter which is placed between the illuminator and the condenser lens blocks most of the light from the illuminator as it passes through the condenser here is that little dark disc here is a normal bright field microscope here is the modified one this opaque disc blocks the light what does that do well only the light that reaches the objective is light that has been refracted or reflected by the structures in the specimen so what results is an image with bright objects on a dark background and here's what you see again dark field microscopy can often create high contrast high resolution images of specimens without using stains this is really nice when you want to view live specimens because live specimens do not like to be stained um because most of the time that kills them and they're no longer alive so if you need to view live specimens oftentimes dark field microscopy is utilized here are some other dark field images again notice the dark background with the light organisms same thing here this is you see a spirulium here is blood um under a dark field microscope phase contrast microscopy phase contrast microscopes use refraction and interference caused by structures in a specimen to create high contrast high resolution images without staining again ideal for living organisms this microscope creates an image by altering the wavelengths of light rays passing through the specimen to create altered wavelengths um wavelength paths an annular stop is used in the condenser the annular stop produces a hollow cone of light that is focused on the specimen before reaching the objective lens the objective contains a phase plate containing a phase ring what does this all do well light traveling directly from the illuminator passes through the phase ring while light refracted or reflected by the specimen passes through the plate this causes waves traveling through the ring to be about one half of a wavelength out of phase with those passing through the plate ultimately structures that differ in features such as refractive index will differ in the levels of darkness what this really does is um you get wave interference the the waves that are you know you'll create that you know you get wave interference where waves that can add together will create bright spots and some waves will actually cancel them cancel each other out creating dark areas so here is an example here is the regular bright field image when you use the phase contrast again it passes through the various parts of the phase contrast microscope creating wavelengths that are half a wave out of sync and some waves will add together in wave interference creating bright spots while others will you know subtract from each other creating dark spots and that really really increases the contrast and the resolution another type of microscope is the differential interference contrast um the dic microscope also known as the normarski optics are similar to phase contrast microscopes and they use interference patterns to enhance contrast between different features of a specimen in a dic microscope two beams of light are created in which the direction of the wave movement polarization differs once the beams pass through either the specimen or the specimen free space they are recombined and effects of the specimens cause differences in the interference patterns generated by the combining of the beams again some areas will be cancelled some areas will be brighter the results you get this really cool image a high contrast three-dimensional image where um and this is all due to the way it comes together these microscopes are especially useful in distinguishing structures within live unstained specimens anytime you can avoid stain um you can probably rest assured that that is going to be good for viewing living organisms again this is just the image of the dic microscopy fluorescence microscopy a fluorescence microscope uses fluorescent chromophores called fluorochromes which are capable of absorbing energy from a light source and then emitting this energy as visible light most of the time the light source that is used is high energy high energy uv light which we cannot see or blue light and then it absorbs that and emits visible light that we can see fluorochromes include naturally fluorescent substances such as chlorophylls as well as fluorescent stains that are added to the specimen to create the contrast the microscope transmits an excitation light generally a form of emr with a short wavelength again that's the uv light or the blue light toward the specimen the chromophores absorb the excitation light and emit visible light with longer wavelengths lower energy longer wavelengths the excitation light is then filtered out in part because ultraviolet light is bad for your eyes um so that only the visible light passes through the ocular lens this produces an image of the specimen in bright colors against the dark background as you can see over here fluorescence microscopes are especially useful in clinical microbiology they can be used to identify pathogens to find particular species within an environment or to find the locations of particular molecules and structures within a cell there's you know when we use it in terms of uh one of the applications is immunofluorescence which is used to identify certain disease-causing microbes by observing whether antibodies bind to them there's two types direct immunofluorescence assay that's dfa indirect immunofluorescence assay that is ifa so in the dfa you see right here in this image the antibody holds the fluorochrome itself and that antibody will bind to the pathogen if it's if the pathogens there it will bind you shine the excitation light on it and whoosh it will emit light in so and this is called a primary antibody stain because the stained antibodies attach directly to the pathogen again the antibody attached to the pathogen is also the one carrying the fluorochrome in an ifa and indirect immunofluorescence you use secondary antibodies that are stained with the chlorophyll chloroform excuse me the fluorochrome and then you so this process um you have an antibody to the pathogen and now you have an antibody to the antibody the antibody contains the secondary antibody contains the fluorochrome so if you you first treat with the primary antibody it binds to the pathogen if it's there and then you treat with a secondary antibody and it will bind to the primary antibody and if it's this pathogen is present now you shine light on it it will light up but it's a two-step process versus the direct which is a one-step process and again just explaining that further here are some more images of from fluorescence microscopy all right so confocal microscopy a confocal microscope uses a laser to scan multiple z planes successively this produces numerous two-dimensional high-resolution images at various depths which can be constructed into a three-dimensional image by a computer confocal microscopes are very useful for examining thick specimens such as biofilms which can be examined alive and unfixed so here is again the uh this is a roof dwelling cyanobacterium biofilm that you see in this picture but again you can visible visualize it alive and directly a two-photon microscope the two-phone microscope uses a scanning technique fluorochromes and long-wavelength lights such as infrared to visualize specimens the low energy associated with a long wavelength light means that two photons must strike a location at the same time to excite the fluorochrome the cool thing about this is that it can be used with living cells with intact tissues um you can do brain slices embryos whole organs and even an entire animal so you can look at something while it's alive and seeing what's going on using this microscope uh the microscope is extremely expensive a single two photon microscope typically costs three hundred thousand to five hundred thousand dollars ufta big price tag all right now we get into electron microscopy electron microscopy um also known as em uses short wavelength electron beams rather than light to increase magnification and resolution and em can produce a sharp image that is magnified up to one hundred thousand x ems can resolve subcellular structures as well as some molecular structures it can see a single strand of dna very cool electron microscopy cannot be used on living material because the methods needed to prepare the specimens it's pretty invasive you'll see here in a moment there are two basic types transmission which is a tem and a scanning electron microscope which is an sem let's first look at the transmission scope uses an electron beam from above the specimen that is focused using a magnetic lens and projected through the specimen onto a detector electrons pass through the specimen hence the transmission electron microscope and that allows the detector to to capture the image for electrons to pass through the specimen in a tem the specimen must be extremely thin which means it must be sliced extremely thin which is typically bad for a living thing so here are some tem images uh just remarkable here is a mitochondria look at the detail now again electrons are passing through this sample so that you can visualize this here is an example of a plant cell tem but again just the detail you know mitochondrias those are small organelles within a cell and and here you get a beautiful image of it with lots and lots of detail scanning electron microscopes form images of the surface of specimens usually from electrons that are knocked off of specimens by a beam of electrons this can create highly detailed images with a three-dimensional appearance that are displayed on a monitor specimens are typically dried and prepared with fixatives that reduce artifacts such as shriveling that can be produced by the drying effect um they're also sputter coated with a thin layer of metal such as gold again not so good for living things and here are some images i love the sem images i just think they're so remarkable here's the head of a fly here are bacteria and here is a small parasite um looks like something that might pop out of your chest and from the alien movie but just crazy details and their 3d images just remarkable and beautiful tem versus sem tm requires thin specimens 20-100 nanometers specimens are enhanced with heavy metal stains you get 2d images max magnification 500 000x while in sem you need to sputter coat with gold um 3d external images are obtained and max magnification 30000x here just some more images notice the 2d nature of the transmission versus the 3d nature of the scanning electron microscope um scanning probe microscopy scanning tunneling microscope the probe passes above the specimen with with both of these types your little probe goes over and it's almost like little lightning bolts and they're detecting things um you know the probe passes above the specimen voltage bias causes electric current between the probe and the specimen intensity is based on distance between the probe and the specimen stm can effectively map the structure of a surface at a resolution where individual atoms can be seen the atomic force microscope thin probe passing above the specimen measures variations in a constant current and afm establishes a constant current and measures variations in the height of the probe tip as it passes over the specimen again individual atoms can be detected over here letter a you'll see you can this is uh the stm of gold and you can see the individual atoms here's an afm of the strand like molecules of nanocellulose those are incredibly tiny and you get some really great images here all right so here is really just a review of all the light microscopes the microscope type the key uses and some sample images you may pause this if you need to look at this continuing electron microscopes again the microscope type the key uses the sample images scanning probe microscopes um one thing didn't mention earlier uh this can get up to 100 million times magnification that is crazy remarkable but again microscope type key uses sample images and that is the end of section 2.3 i hope that you are now able to identify and describe the parts of a brightfield microscope i also hope that you are able to calculate total magnification for a compound microscope and describe the distinguishing features and typical uses for various types of light microscopes electron microscopes and scanning probe microscopes thank you very much for tuning in mr luther out