Outcome based observations (at high objective magnitude) of various fixatives led us to conclude that less than 10% formalin best preserves detail, keeping the tissue intact and similar to its natural state in the body the shrinkage is decreased, providing accurate diagnostic material.

Over 600 experiments were conducted on various tissues with assorted fixatives in order to reach this conclusion.

Why use 10% neutral buffered formalin when its osmolarity is nearly 10X the inside of a normal cell?  Our fixative, with outcome based detail, is less than half that. What’s the first thing that happens at the tissue interface when its put into 10% phosphate buffered formaldehyde?  Answer:  The osmotic difference causes water to come out and shrink the tissue when formaldehyde diffuses in.  This shrinkage causes the compaction of the structural and other proteins in the cell so the cross-linking methylene bridges are denser and overdone.  

Osmolarity is an important consideration when analyzing fixation, and is defined as a measure of the osmoles of solute per liter of solution.  The osmolarity of a solution can be calculated with the following formula (below information from Wikipedia):

Osmol/L = Σ φi ni Ci
•    φ is the osmotic coefficient, which accounts for the degree of non-ideality of the solution. In the simplest case it is the degree of dissociation of the solute. Then, φ is between 0 and 1 where 1 indicates 100% dissociation. However, φ can also be larger than 1 (e.g. for sucrose). For salts, electrostatic effects cause φ to be smaller than 1 even if 100% dissociation occurs (see Debye-Hückel equation);
•    n is the number of particles (e.g. ions) into which a molecule dissociates. For example: glucose has n of 1, while NaCl has n of 2;
•    C is the molar concentration of the solute;
•    the index i represents the identity of a particular solute.

The equations involved with the vapor phase of formaldehyde and dissolved formaldehyde, along with the reversibility of formaldehyde methylene bridges and dissolved formaldehyde, still operate in tissue sections (deparaffinize a block and smell).

Note:  The ionic strength of 10% neutral buffered formalin allows for the precipitation of proteins by van der Waal forces, which clouds results of proper fixing and cannot be recovered.  The ionic strength causes proteins to unfold, allowing the van der Waal forces to take over.  Charged shielding also plays a role in allowing irreversible hydrophobic precipitation to take place (van der Waals).  The DLVO theory is connected to the precipitation which occurs in 10% neutral buffered formalin (for more information go to Wikipedia and http://www.malvern.co.uk/LabEng/industry/colloids/dlvo_theory.htm).

   Standard 10%    Ultralight   Standard 10%  Ultralight

   Bladder                      Prostate

VALIDATION

 Due to the accurate detail our process fixative produced, a “new” cell feature was observed: the fact that cells exist in post mitotic pairs, where one nucleus is bigger and chromatin is clumped differently.  The electron micrograph light/dark cells used to be explained as “artifact with no explanation”.  Over 35,000 photomicrographs consisting of a phylogenetic trace of this new cell feature have been carefully studied and documented, suggesting that cells exist in post mitotic pairs.  Non- siliconized diatoms were observed: mosses, mushrooms, fungi, amoebas, sponges, spiders, insects, snails, slugs, round and flat worms etc.  This extensive phylogenic comparison validates our system of histology and opens endless possibilities for advancement in biology due to an updated and accurate tissue processing system.

Slug Ovum (light/dark cells)     Human Cartilage (light/dark cells)     Human Ova (light/dark cells)
   
Proof of Asymmetry:
1.  Biochemistry: asymmetrical methylation occurs at the replication fork of DNA in original and daughter strands (paracentromere non-coating transposon region). More specifically, the terminal tails of the histones of daughter cells are differentially methylated at location K16.  Daughter cells have 2000-3000 genes expressed differently suggesting a functional link to this asymmetry.
2.  The difference in staining was differentially checked with a spectral microscope that showed a reflective wavelength shift in mitotic figures (two different colors), verifying the observable difference noted in the nucleus after the Ultralight process of histology (Carnegie Mellon University).
3.  Clones of lymphocyte were taken to a flow cytometry lab, where Gaussian distribution was shown to be a summation of two Gaussian distributions, with the peak shifted as a result of gating, where the two different populations of daughter cells (CLL lives used) were separated.  This resulted in two chromatin detail populations on separate smears.

Further Validation by Mother Nature

Molluscum Contagiosum

During our histologic trace for asymmetric chromatin on skin biopsy slides, it was observed that the asymmetric chromatin in squamous epithelium facilitated the asymmetric expression of molluscum contagiosum viral particles in only the darker staining chromatin pattern cells.

FORMALIN PIGMENT

 Formalin pigment has only been observed on one Ultralight prepared slide of millions over the past fifteen years.  We hypothesize that formalin pigment arises from the phosphate buffer related charge density and a van der Waals hydrophobic interaction.  We have no phosphate buffer in the Ultralight process.

DEHYDRATION

 The use of absolute alcohols (95% ethyl, 5% methyl, 5% isopropyl) with ethyl alcohol at 95% will dilute water in tissues by miscibility- it does not dehydrate.  This incomplete dehydration helps produce artifacts and impairs detail.  Dehydration, even with absolute alcohol, is not complete dehydration.  There are residual dedicated water molecules associated by dipole layers to some proteins and protein structural water remains.  Absolute alcohol is used in the Ultralight process as it yields the best results, keeping tissue structure intact.

THICKNESS

 1µm is recommended to eliminate the blurred results a depth of field can create.  We can cut down to .5µm for detail localizing within mitochondria, or up to 10µm. 

Blue: Dapi nuclei

Orange: Membrane Porin

Green: Inner membrane oxphos complex

 The temperature throughout the Ultralight process does not exceed 39°F (except paraffin embedding and oven drying).  This conclusion was drawn by outcome based results.

Too many tissues are unnaturally damaged as a result of osmolarity shrinkage.  Heating to quicken the process of fixation unnecessarily ruins detail and preservation of tissue (cooking).

The use of microwaves or ultrasound involves interactions between proteins with focal resonant frequencies, heating and cooking the tissue to cause irreversible artifacts.  Think of temperature in relationship to induction of heat shock proteins- Mother Nature will give the answer- heat unfolds proteins and disrupts cytoskeletal architecture.

ANTIGEN RETRIEVAL

 For a more holistic understanding of antigen retrieval, one must look at protein sequencing, protein folding, phagocytosis, acid hydrolase digestion, antigen presentation and antibody editing.  

Often, the antigen sequence is not seen on the surface, thus the need for antigen exposure is necessary.  The folding of proteins is thermodynamically determined by protein sequence and hydrophilic and hydrophobic interactions.  Most antigen exposure relies on basic protein biophysics of unfolding, not so much on the reversing of formalin fixation.  Some of antigen retrieval will come from methylene bridge reversal, but the majority depends on the entire protein molecule being unfolded in varying and often extreme physical chemistry (this is antigen exposure not antigen retrieval from formalin based processing).  

This discussion points to some extreme measures taken in the field (to heat tissue or lower or raise pH) that ultimately results in the degradation of tissue detail, as antigen exposure is not selective.  Choosing the route for the least destruction of detail is vital.

Cytokeratin Fibers in Keratinocytes

 We have taken time to detect the fact that formaldehyde, hematoxylin, and eosin all react with the same amino acids.  The literature explaining the binding of hematoxylin to the phosphate skeleton of DNA goes back to 1954, some years before Watson and Crick elucidated the double helix of DNA and later, their structuring of histone octomers wrapped with DNA (the nucleosome).  Incubating fresh tissue in buterate or tricostatin (histone deacetylase inhibitors) results in no stain of the nucleus by hematoxylin.  The standard explanation for where hematoxylin binds is obviously incorrect.  Histone octomers by themselves do not stain (chicken histone octomers obtained from Oakridge National Laboratories/ University of Tennessee).  It is theorized that hematoxylin is binding to the histone tails with the appropriate amino acids in a type of intercalative interaction.  Eosin appears to bind to proteins with the same positively charged amino groups as formaldehyde does.  These are great examples of differential competitive chemistry and biophysics.  

DIAGNOSTIC PATHOLOGY

Prostate- Vastly improved cytologic detail

 The extra clarity in detail of the Ultralight process does not alter the established classification in surgical pathology.  It simply refines the process of diagnosis, making it more accurate and efficient.

WHAT GOOD IS THIS METHOD?

ER in pancreas

Endoplasmic Reticulum
We can resolve profiles of membranes of endoplasmic reticulum when perpendicular orientation is present.  This is a tool for research.

Golgi in colon

 Protein Folding Disorders
We have the ability to look between the membranes of golgi in some cells.  This is due to our preservation of detail and superior EM referenced localization.  This is another tool for research and later diagnosis. 

Mitotic Spindle & Microtubules

Mitoses
We are currently judging our histology and staining by how well we resolve individual chromosomes.  We have many examples to demonstrate chromatin staining asymmetry in cell division from an extensive phylogenetic trace.  What are the implications of this in cancer?  Our observations over the past fifteen years note this asymmetry in various cancers without exception when the case is carefully studied (1000-1600X).  What does this mean for cell biology?  The biochemistry of this asymmetry was partially presented at the 2008 Abcam meeting in Antigua in Arabidopsis (Barbara McClintock).  This is asymmetric methylation of cytosine on the original strand of DNA at the non-coding pericentromeric transposon region and not on the daughter strand.  Subsequent methylation of lysine residues translate into different chromatin condensation and subsequently hematoxylin staining.

Economics
A good high-resolution optics light microscope is around $50,000.

A confocal microscope with inferior detail is about $500,000.

An electron microscope is out of the question.

A SPIM microscope for fluorescence is no longer needed.  We have single plane fluorescence down to .5μm.  

There are multiple ways to quench background fluorescence.  We can eliminate it with simple software.

Our Ultralight Histology process takes no more time, no more effort, no significant increase in cost, and is using the same fundamental pathway that was developed a century ago.  With our method, it is just as easy to cut at 1μm as it is at 4μm or 5μm.

Cancer
Pathologists diagnose and classify malignancies.  Classification schemes were developed over 100 years to give indication of prognosis and behavior of tumors.  Current chemotherapy is data driven by studies that have been done with treatment trials using this classification.  The drug development is currently focused on molecular pathways that are aberrant in different cancers.  A large genomic study on fifty different cancers with genomes, determined on 200 cases, is beginning to define multiple different pathways to produce a neoplasm in a single classification entity.  As drugs emerge, it will be important to provide testing on particular neoplasms with unique abnormalities.  So far, early results suggest that some cancers may have in the neighborhood of twenty different pathways to the same morphologic classification.  

Now if you were a cancer patient, you would not be as interested in classification and prognosis as you would in how the doctors will treat the cancer - for cure or control?  If we redo the oncology studies the same way we did in the past, this type of answer may be decades in the future.  We suggest that the current technology will soon allow for multi-fluor multiplexed fluorescence testing of these many pathways on one slide.  Current software and hardware now available is at the level of eight different photofluors multiplexed in one test.  The scale up is not that far into the future.  

Breast Nuclear Pores

There are two obvious requirements of histology; that the details are optimized and the background fluorescence be minimized.   These requirements are possible with the Ultralight Histology process with our slides that are .5μm thick with very little background fluorescence.

We propose that down the road, with all the new receptor blockers and pathway inhibitors in drug development, that this is a needed expertise that can be performed on histologic specimens.  CANCER PATHWAYS PROFILE TESTS ON ONE SLIDE!

Obesity Related Steatosis
There is an increase in steatosis diagnosis related to obesity.  If you are a patient and you had a liver biopsy, you would get a classification of your obesity with no suggestion of why you have it and no suggestion of any treatment.  Most steatosis is related to mitochondrial functions.  The Ultralight process has allowed Bi-Biomics to be the first to do intra-mitochondrial localization using fluorescent dyes.  From what we have done at this point, it is apparent that mitochondrial function varies from zone to zone.  There are over fifty antibodies available from Mito-Sciences.  It would make sense to provide multiplexed multi-fluor fluorescence on one slide to indicate what abnormalities are related to the steatosis.  Keep in mind mitochondrial proteomics and functions are very complex.  Minor mitochondrial dysfunction is likely to be relatively common (only 15% of individuals that are major league alcohol consumers – a liter a day- develop chirrosis).  From the mitochondrial literature and meetings we know there are supplements that can decrease the disease effects in some patients.  Can these be used in steatosis?  Can future developments in treating mitochondrial abnormalities be used in steatosis?

Cell Biology
The current tools for life sciences involving cells, uses fluorescent microscopes with time-lapse photography.  This new tool has allowed researchers to look at cells in real time and the phenomena of binary cellular behavior is beginning to emerge.  That is the behavior of cells in the culture demonstrating interactive features of cells as pairs (biophysics meeting in 2008 - presentation on the biophysics of the immune response).  Watch for further developments in this area.

In electron microscopy, there is a misunderstood phenomenon of light and dark cells that has never been explained and is ignored as a “possible artifact”.  However, when the Ultralight process was developed, and the comparisons with electron micrographs were made at 1000X, the light and dark cells were brought into light microscopy.  A histologic trace was done on human tissue and samples were taken from many sources for a phylogenetic trace.  We found NO examples of any organism that did not display this light and dark feature.  We feel we have explained the light and dark cell phenomena in asymmetrical cell division.  Exactly what light and dark cells mean biologically is a mystery on our back burner.  We have observed these two cell types to form patterns in some tissues- this is another layer of complexity in cell biology that appears universal and currently has no explanation.  We refer to this biologic function as binary mesobiology.  Stay tuned and watch it evolve in cell biology research!

Binary MesoBiology
With unpublished work done over fifteen years, and over 50,000 documented images, we have observed what we refer to as binary mesobiology as an organization of metazoans.  The size quarter is .2-2μm for observable detail.  The organisms are assembled with post-mitotic pairs that have asymmetric chromatin and gene expression, have spatial entanglement with adhesion molecules, and are by some process, temporally entrained.  Much of this conclusion has also been observed on a tissue culture scale by Jan Ellensburg Group at the European Molecular Biology Lab in their knock down studies on mitosis.  Publications are intended.

STANDARD HISTOLOGY AS A 100 YEAR OLD METHOD WAS DEVELOPED IN A PERIOD OF TRANSITION FROM RESIDUAL ALCHEMY TO MODERN CHEMISTRY.  THIS ANCIENT METHOD CAN NOT BE SCIENTIFICALLY DEFENDED AND HAS NEVER BEEN VALIDATED WITH PRESERVATION OF DETAIL AT 1000X TO COMPETE WITH PUBLISHED ELECTRON MICROGRAPHS.  

WHAT METHOD ARE YOU USING?  WHAT IS YOUR VALIDATION?  


FIRST PUBLICATION OF OUR “92 THESES” OF ULTRALIGHT HISTOLOGY ON THE DOOR OF THE CATHEDRAL “STANDARD HISTOLOGY METHOD”

Sources include: Biophysical Society, Society for Biochemistry and Molecular Biology, American Society of Cell Biology, Protein Society, Matrix Society, Mitochondrial Meetings, Genome Meetings, Systems Biology Meetings, Abcam and other Chromatin Meetings, Drug Development Meetings and numerous presentations and posters (membership and meeting attendance to the above listed).