Live Cell Imaging

Ideal Incubation Conditions

Cells and tis­sues cul­ti­vat­ed in vit­ro sim­ply need the same con­di­tions as they have in vivo. Eas­i­ly said and easy to under­stand, it is often dif­fi­cult to achieve this on a micro­scop­ic set­up. With our long-term expe­ri­ence in micro­scop­ic cell and tis­sue cul­ti­va­tion, we have devel­oped solu­tions that are both easy to use and give the cells the best pos­si­ble, most homo­ge­neous con­di­tions in every incubation-relevant para­me­ter.

Temperature

Tem­per­a­ture is the pri­ma­ry para­me­ter for a suc­cess­ful in vit­ro cell cul­ti­va­tion. If the tem­per­a­ture is not like in vivo, a dif­fer­ing phys­i­o­log­i­cal behav­ior will occur very rapid­ly. Cur­rent sci­en­tif­ic research is ongo­ing in this field, and a lot of tem­per­a­ture sen­si­tive pro­teins have already been iden­ti­fied. Since the first obser­va­tion of the expres­sion of spe­cif­ic pro­teins was under heat stress, they are gen­er­al­ly called “heat shock pro­teins”.

A pre­cise, sta­ble and, in par­tic­u­lar, homo­ge­neous heat­ing of cell incu­ba­tion ves­sels (espe­cial­ly when using multi-well spec­i­men) is thus a major fac­tor for a suc­cess­ful cell obser­va­tion on the micros­cope. This is one of the main tar­gets in the devel­op­ment of our pro­ducts.

Two dif­fer­ent buzz­words for the two dif­fer­ent approach­es of main­tain­ing the tem­per­a­ture of cell cul­ture ves­sels on the micros­cope are found in the web: “Stage Top Incu­ba­tor” for small incu­ba­tion inserts that are just put into the stage open­ing and “Cage Incu­ba­tor” for large cham­ber incu­ba­tors, enclos­ing most parts of the micros­cope. We at PeCon do not use  the term “Cage Incu­ba­tor”, because in our opin­ion your cell and tis­sue cul­tures must not be kept in a “cage” to pre­vent them from escap­ing.

PeCon offers a large selec­tion of stage-top incu­ba­tion pro­ducts and acces­sories to enhance system’s per­for­mance for a pre­cise, sta­ble and uni­form heat­ing of spec­i­men.

The “gold stan­dard” for live cell imag­ing on the micros­cope is a large cham­ber incu­ba­tor, that heats not only the spec­i­men, but all micros­cope parts which are in con­tact with your sam­ple. Addi­tion­al­ly, mechan­i­cal drifts in the X, Y or Z-Axis are min­i­mized, because the ori­gin of such a mate­r­i­al expan­sion of stages etc. by chang­ing tem­per­a­tures is reduced.

A large cham­ber incu­ba­tor is also the basis of a very ver­sa­tile and upgrade­able incu­ba­tion sys­tem. Trans­par­ent, light tight and laser-safe incu­ba­tors are avail­able.

pH value (CO2)

The opti­mal pH val­ue for most cell lines is 7.4, but can be slight­ly dif­fer­ent from cell type to cell type. Cell cul­ti­va­tion media com­prise glu­cose, salts, amino acids, vit­a­mins, oth­er sub­stances and a buffer sub­stance to bal­ance the pH val­ue.
HEPES is a so-called “sta­ble buffer”, it also main­tains the pH val­ue at ambi­ent air con­di­tions, how­ev­er, this sub­stance influ­ences the cell behav­ior neg­a­tive­ly. Even pho­to­tox­ic effects are report­ed, which exclude its usage for micro­scop­ic cell obser­va­tion.
The phys­i­o­log­i­cal buffer is NaH­CO3, because it is the sub­stance in most organ­isms to bal­ance the pH val­ue in blood and tis­sues. For this rea­son, NaH­CO3 is used to buffer the pH of com­mon cell cul­ture media (like MEM, DMEM, RPMI 1640). Since this sub­stance can dis­so­ci­ate into H2O and CO2, and the lat­ter can just escape into the ambi­ent air with its low CO2 con­cen­tra­tion, the buffer is con­sid­ered as “not sta­ble”.

Relationship between CO2 concentration and pH value in nutrition media
Rela­tion­ship between CO2 con­cen­tra­tion and pH val­ue in nutri­tion media

There­fore, the CO2 con­cen­tra­tion in the atmos­phere around the cul­ture media has to be increased, until a steady state con­di­tion is achieved, where the same amount of CO2 escapes from and dis­solves into the media. Depend­ing on the NaH­CO3 con­cen­tra­tion used in media for­mu­la­tions, a CO2 con­cen­tra­tion of 5.0% (2.2 g/l) or 7.5% (3.7 g/l) is need­ed.

A sta­ble CO2 con­cen­tra­tion ensures a sta­ble pH in the nutri­tion media. Again, a homo­ge­neous dis­tri­b­u­tion in the incu­ba­tion cham­ber is vital for mult well spec­i­mens. Our gas con­trollers, togeth­er with our small incu­ba­tors or local CO2-Covers, gen­er­ate this sta­ble and uni­form CO2 con­cen­tra­tion. All con­trollers work with stan­dard low-priced tech­ni­cal CO2 and do not need an addi­tion­al com­pressed air sup­ply. An in-house devel­oped CO2 sen­sor ensures a fast and reli­able mea­sure­ment with a large detec­tion range of 0-20% and a high res­o­lu­tion of 0.01%. It is specif­i­cal­ly designed for cell cul­ti­va­tion needs, and is the basis for a quick and sta­ble CO2 con­trol with our con­trollers.

Oxygen (o2)

Cells under in vivo con­di­tions are nor­mal­ly not exposed to the high oxy­gen lev­el found in our atmos­phere (20.8%), but are usu­al­ly at an oxy­gen con­cen­tra­tion of 10-13% (human blood) or low­er. It is also known, that some dif­fer­en­ti­a­tion process­es like red blood cells from stem cells (ery­thro­poiesis) only occur under low­er oxy­gen con­cen­tra­tions. High­er oxy­gen con­cen­tra­tions are the trig­ger for the migra­tion of spe­cial cells for per­form­ing wound heal­ing.

Besides that, oxy­gen is a bi-radical, react­ing very eas­i­ly with oth­er mol­e­cules and, thus, mod­i­fies them. Oxida­tive stress can lead to neu­rode­gen­er­a­tive dis­eases like Parkinson’s dis­ease or Alzheimer’s dis­ease. Oxy­gen also dam­ages the DNA and cells have to per­form elab­o­rate DNA repair mech­a­nisms to main­tain their genome sta­bil­i­ty.

In order to offer cells the best pos­si­ble con­di­tions sim­i­lar to in vivo, not only in the lab­o­ra­to­ry incu­ba­tor, but also on the micros­cope, a reduc­tion of the oxy­gen con­cen­tra­tion should be con­sid­ered. Our gas con­trollers for oxy­gen use an oxy­gen sen­sor with a very long life­time and can reduce the oxy­gen con­cen­tra­tion from ambi­ent 20.8% down to 0.1% by a dis­place­ment with nitro­gen. Due to their fast response times on set­point changes, they can also be used for dynam­ic exper­i­ments.

Humidity

Evap­o­ra­tion of water out of the cell cul­ture media hap­pens every time when the rel­a­tive humid­i­ty (rH) in the atmos­phere around the cell cul­ture ves­sel is <100%. How­ev­er, most ves­sels have lids with only small gaps, so the dry­ing effect is reduced, thus not elim­i­nat­ed. But cul­ti­va­tion times of sev­er­al hours with­out a humid­i­fi­ca­tion of the atmos­phere is pos­si­ble with­out any neg­a­tive effects on cell behav­ior or vital­i­ty.

The rel­a­tive humid­i­ty is ref­er­enced to the amount of water that the air can hold at a giv­en tem­per­a­ture. The high­er the tem­per­a­ture, the more water can be absorbed. There­fore, when heat­ing up the ambi­ent air at 22°C with a rel­a­tive humid­i­ty of 60% to 37°C, the rel­a­tive humid­i­ty drops down to ~25%. While at 22°C only 19.4 g/m³ water is need­ed to have 100% rH, at 37°C already 43.8 g/m³ of water (more than twice the amount) is nec­es­sary.

Inside a large incu­ba­tor, a humid­i­fi­ca­tion is easy. Just place the humid­i­fi­ca­tion bot­tle that is sup­plied with every PeCon gas con­troller and the sup­ply tub­ing to the CO2-Cov­er inside the heat­ed incu­ba­tor. Physics will do its job and you will obtain a very effec­tive humid­i­fi­ca­tion of the atmos­phere around your spec­i­men. For stage top sys­tems, we have an exter­nal heater for the humid­i­fi­ca­tion flask. Com­bined with an iso­lat­ed, heat­ed tube, this is also very effec­tive against the evap­o­ra­tion.
Since most cell cul­ture media have to be changed after 2-3 days because they have been used up by the cells, this nec­es­sary media change resets the neg­a­tive effects of evap­o­ra­tion. If,  with­in this peri­od of time, no harm­ful evap­o­ra­tion takes place, there is no need for exten­sive mea­sures against the evap­o­ra­tion.

PeCon offers two dif­fer­ent approach­es to reduce the evap­o­ra­tion which can also be com­bined:

  1. Seal­ing the cul­ti­va­tion ves­sel with water­tight but gas-permeable foils (Foil­Cov­ers)
  2. Increas­ing the humid­i­ty around the spec­i­men (local humid­i­fi­ca­tion)