Biological Sample Preparation

Electron microscopy is a crucial technique used in the life sciences to visualize microstructures that would otherwise be too small to observe. Scanning electron microscopy (SEM) can reveal topographical information about biological samples such as the shapes of peripheral proteins on cell membranes. Transmission electron microscopy (TEM), though requiring significantly more preparatory work, provides information about structures within the interiors of biological samples, such as the shapes of organelles within a cell. As an advanced imagery technique, electron microscopy (EM) requires advanced sample preparation techniques in order to prevent damage to both the sample and the electron microscope, especially in the case of biological samples.

Dangers of Imaging Unprepared Biological Samples

There are several obstacles in the way of having an unprepared biological sample, such as a recently dead insect, being imaged under an electron microscope. These obstacles result in 3 main issues: poor image quality, damage to the EM instrument, and damage to the sample.

Outgassing

Outgassing occurs when an object releases trapped gases when placed in a vacuum, resulting in damaging or contamination of the vacuum components. All unprepared biological samples will have some air in them, typically dissolved in some fluid which permeates a tissue. When a vacuum is pulled, these gases rapidly expand, potentially damaging equipment by evaporating or even sublimating contaminants, and potentially damaging the sample by increasing its temperature and ripping apart tissues due to the expansion of its gases. These contaminants will interfere with vacuum seals and can even get 'kicked up' (sucked upwards in a drift of gases) into machine components like apertures (thin strips of metal meant to regulate the electron beam). Meanwhile, the sample will be destroyed due to heat denaturing proteins and gas expansion ripping through microstructure.

Moisture

All unprepared biological samples will have some amount of moisture in them. Liquids such as water can evaporate and contribute to outgassing, but can also create a humid environment within the sample chamber resulting in contamination. Water vapor can then get in the path of electrons and deflect/eject them in unpredictable directions, resulting in lesser image quality and damage to the instrument.

Conductivity

Most unprepared biological samples will be nonconductive or poorly conductive. This results in poor image quality and charging of the sample, which can heat it up and degrade it.

Degradation

Biological samples contain molecules that can be quite fragile under the intense barrage of an electron beam. Proteins are made of amino acid chains with various bonds including hydrogen bonds, hydrophobic interactions, disulfide bridges, and ionic bonds. High speed electrons can break these bonds, resulting in the denaturing of the protein and damaging of the structure that these proteins make up. Furthermore, biological samples tend to get moldy over time, obscuring and damaging microstructure and creating biohazards.

Obstacles with Preparing Biological Samples

Biological samples typically require special preparation since the conventional means of preparing samples for EM can damage their fragile microstructures. Removing a biological sample's moisture through means of vacuum desiccation will result in the sample expanding and ripping apart. Heating up a sample in hopes to evaporate the moisture can potentially burn it and likely won't get all of the moisture out. In fact, any non-chemical means of removing moisture will likely be ineffective.

Procedure

Multicellular Organic Samples (Ex: Insects)

• Dessication process using Ethanol baths and HMDS
  • Using vacuum drying will deform sample and allow degradation
  • Time estimate: ~1 hr working inside MILL + ~1 day waiting
  • Source: https://app.jove.com/v/20111/chemical-drying-biological-sample-preparation-for-scanning-electron

1. Sample fixation using fixative and pH buffer
   1. To prevent degradation of cell structure and denaturing of molecules
   2. Place sample in small centrifuge tube (~1.5 mL or higher depending on sample size; ensure complete submersion of sample in solvent is possible)
   3. OPTIONAL: Completely submerge samples in fixative for 2 hours at 4℃
      1. Only worth doing if you have the equipment, are following safety precautions, and have a sample that absolutely necessitates chemical fixation (otherwise it will very quickly degrade)
      2. Types of Fixatives (source):
         1. Aldehydes
            1. Formaldehyde, glutaraldehyde
            2. Extremely dangerous - carcinogenic, toxic fumess, bad for environment, flammable
            3. Requires fume hood and proper safety gear
            4. Less sample degradation compared to using alternative fixatives
         2. RECOMMENDED: Organic Fixatives
            1. Ethanol, methanol, acetone
            2. Flammable, but less dangerous than aldehydes
            3. Requires slow introduction of fixative to the sample to prevent deformation
               1. Slowly submerging sample in increasing concentrations of organic fixative
               2. Instructions for using ethanol below
         3. Sample-Specific Fixatives
            1. Carnoy’s Solution, Helly’s Solution, Zinc Fixative
            2. Depends on what specific sample you are viewing with SEM
            3. Research safety and protocol
   4. After submerging sample in fixative for 2 hours at 4℃, remove fixative with disposable pipette
   5. OPTIONAL + RECOMMENDED: Submerge sample in pH buffer solution
      1. Submerge sample in ~1mL phosphate buffer solution pH 7.2 for 10 minutes @ room temp (phosphate buffer solution pH 7.2)
      2. Remove buffer with pipette
   6. If using ethanol for fixation, follow procedure below:
      1. Submerge sample in 25% ⇒ 50% ⇒ 75% ⇒ 100% ethanol solution for 10 minutes each (remove solution with pipette after each 10 minutes)
   7. REQUIRED: Chemical Desiccation
      1. Methods of desiccation:
         1. HMDS
            1. Acute dermal toxicity (SDS), requires proper PPE
            2. Submerge with 1:2 solution of HDMS & 100% ethanol 20 minutes, remove w/ pipette
            3. Submerge with 2:1 solution of HDMS & 100% ethanol 20 minutes, remove w/ pipette
            4. Submerge with HDMS 20 minutes, remove w/ pipette
            5. Repeat c-e once
            6. Let sample dry in fume hood after barely submerged with HMDS (place roofing over sample to prevent anything from falling into it
         2. Silica Gel
            1. Significantly less dangerous, solid, nonflammable powder that can irritate skin via dehydration and should not be consumed
            2. Cover sample with pellets and place in airtight container (plastic zip bag will suffice) for 12-24 hours
2. Place desiccated sample onto aluminum stub with carbon/copper tape, blow gently with compressed air
3. RECOMMENDED: Providing a conductive pathway for electrons
   1. 2 Methods:
      1. Sputter coating
         1. Either Gold, Palladium, or Carbon sputtering (Gold recommended)
         2. Will remove some of the more intricate surface details from your sample
         3. Follow SOP for coaters
      2. Using conductive paint (silver/carbon)
         1. Will add ~3 hours to wait time
         2. Paint the outside ring of the aluminum stub with the conductive paint
            1. The purpose of this is to give the electrons which are being shot into the sample somewhere to go; we will be connecting the nonconductive sample (which is being bombarded with electrons) to the conductive aluminum stub (which is more suited to hold those electrons) via the conductive paint
            2. After painting the outside ring of the aluminum stub with conductive paint, paint some connections towards and just touching your sample - do not paint over portions of your sample you wish to image in detail, just paint enough so that the electrons have a path to follow
         3. Place the aluminum stub with the paint and your sample in a desiccator for 3 hours so that the paint can dry
            1. An airtight container with silica gel will suffice as a desiccator
         4. Once ready to image, blow again gently with compressed air and put into the SEM

Silica gel holds 30-40% of its weight in water via adsorption (water forms a thin layer on surface of silica gel rather than being absorbed within it)

Regenerage by heating up to 120 C for 1-2 hours

• Moisture analyzer